Glycotargeting therapeutics

ABSTRACT

Several embodiments of the present disclosure relate to therapeutic compositions configured to target the liver of a subject and that are useful in the treatment or prevention of one or more of transplant rejection, autoimmune disease, food allergy, and immune response against a therapeutic agent. In several embodiments, the compositions are configured to target the liver and deliver antigens to which tolerance is desired. In several embodiments, the compositions are configured for clearance of a circulating protein or peptide or antibody associated with one or more of the above-mentioned maladies. Methods and uses of the compositions for induction of immune tolerance are also disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/925,628, filed Mar. 19, 2018, which is a continuation ofInternational Application No. PCT/162016/001411, filed Sep. 16, 2016,which is a continuation-in-part of U.S. patent application Ser. No.15/185,564, filed Jun. 17, 2016, which is a continuation-in-part of U.S.patent application Ser. No. 14/859,292, filed Sep. 19, 2015, eachentitled “GLYCOTARGETING THERAPEUTICS.” This application is acontinuation of U.S. patent application Ser. No. 15/925,628, filed Mar.19, 2018, which is a continuation of International Application No.PCT/162016/001411, filed Sep. 16, 2016, which is a continuation-in-partof U.S. patent application Ser. No. 14/859,292, filed Sep. 19, 2015,each entitled “GLYCOTARGETING THERAPEUTICS.” This application is acontinuation of U.S. patent application Ser. No. 15/925,628, filed Mar.19, 2018, which is a continuation-in-part of U.S. patent applicationSer. No. 15/185,564, filed Jun. 17, 2016, which is acontinuation-in-part of U.S. patent application Ser. No. 14/859,292filed Sep. 19, 2015, which is a continuation-in-part of U.S. patentapplication Ser. No. 14/627,297 filed Feb. 20, 2015, which claims thebenefit of U.S. Provisional Application No. 61/942,942 filed Feb. 21,2014, each entitled “GLYCOTARGETING THERAPEUTICS.” The entirety of eachof the foregoing applications is hereby incorporated by reference.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

The claimed invention was made by, on behalf of, and/or in connectionwith one or more of the following parties to a joint research agreementbetween: Ecole Polytechnique Federale de Lausanne (EPFL) and Anokion SA.The agreement was in effect on and before the date the claimed inventionwas made, and the claimed invention was made as a result of activitiesundertaken within the scope of the agreement.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isANOK.002P4C1_ST25.TXT, the date of creation of the ASCII text file isSep. 24, 2020, and the size of the ASCII text file is 49.1 KB.

BACKGROUND Field

Several embodiments of the invention disclosed herein relate topharmaceutically acceptable compositions that are useful in thetreatment of transplant rejection, autoimmune disease, allergy (e.g.,food allergy), and immune response against a therapeutic agent.

Description of Related Art

Various approaches have been used to induce tolerance to antigens thatelicit an unwanted immune response. Some approaches employ targeting ofthe antigens to specific cells. Applications US 2012/0039989, US2012/0178139 and WO 2013/121296 describe the targeting of antigens toerythrocytes to take advantage of the erythrocytes' role in antigenpresentation for tolerization.

SUMMARY

Notwithstanding the positive results generated to date usingcell-targeting approaches, the possibility of alternative approaches hasremained of interest. In particular, several embodiments disclosedherein relate to compositions configured to target one or more celltypes in the liver and, as a result, deliver an antigen to whichtolerance is desired to the one or more cell types targeted, and therebyinduce a processing of the antigen and induce immune tolerance to theantigen. The antigen, as disclosed in more detail below, can comprise atherapeutic agent, a protein, a protein fragment, an antigenic mimic ofa protein or protein fragment (e.g., a mimotope). Additional types ofantigens are discussed in more detail below. Methods and uses of suchcompositions are also provided for, in several embodiments.

In several embodiments, there is provided a compound comprising Formula1:X

Y—Z]_(m)   Formula 1wherein m is an integer from about 1 to 10, X comprises moleculecomprising an antigenic region, Y is of a linker moiety having a formulaselected from the group consisting of:

wherein the left bracket “(” indicates a bond to X, the right or bottombracket and “)” indicates the bond between Y and Z, n is an integer fromabout 1 to 100, where present p is an integer from about 2 to 150, wherepresent q is an integer from about 1 to 44, where present R⁸ is CH₂ orCH₂—CH₂—C(CH₃)(CN); and where present R⁹ is a direct bond orCH₂—CH₂—NHC(O), and Z comprises a liver-targeting moiety. In severalembodiments, X is a protein or protein fragment comprising an antigenicregion. In several embodiments, Z is galactose, while in someembodiments Z is glucose. In several embodiments, Z is galactosamine,while in some embodiments Z is glucosamine. In several embodiments, thealpha anomer of glucose or galactose is used in the composition. Inseveral embodiments, the beta anomer of glucose or galactose is used inthe composition. In several embodiments, mixtures of the alpha and betaanomers are used, including optionally mixtures of glucose andgalactose. In several embodiments, Z is N-acetylgalactosamine, while insome embodiments Z is N-acetylglucosamine. In several embodiments, thealpha anomer of glucosamine or galactosamine is used in the composition.In several embodiments, the alpha anomer of glucosamine or galactosamineis used in the composition. In several embodiments, mixtures of thealpha and beta anomers are used, including optionally mixtures ofglucosamine and galactosamine. Likewise, in several embodiments thealpha anomer, the beta anomer, or combinations of the alpha and betaanomers of N-acetylgalactosamine or N-acetylglucosamine. Combinations ofany of the alpha or beta anomeric forms of any of the liver targetingsugars, and any combinations of the sugars can be employed in variousembodiments. In several embodiments Y comprises Y_(m) or Y_(n), m isbetween 1 and 5, n is between 75 and 85, p is between 85 and 95, and qis between 2 and 6. In several embodiments m is between 1 and 3, n is79, p is 90, and q is 4. In several embodiments, X is selected from thegroup consisting of insulin, proinsulin, preproinsulin, gluten, gliadin,myelin basic protein, myelin oligodendrocyte glycoprotein andproteolipid protein, Factor VIII, Factor IX, asparaginase, uricase andfragments of any of the preceding. In several embodiments, the antigen Xis not a full length protein. For example, in some embodiments, theantigen is not full length gliadin, insulin, or proinsulin. In severalembodiments, the antigen X is not a fragment of a protein. In severalembodiments, m is not greater than 3, n is not greater than 80, p is notgreater than 100 and q is not more than 5.

In several embodiments, Y is a linker moiety having a formula of:

In several embodiments, Y is a linker moiety having a formula of:

In several embodiments, Y is a linker moiety having a formula of:

In several embodiments, Y is a linker moiety having a formula of:

In some embodiments, combinations of the linkers disclosed herein may beused, just as combinations of the liver targeting moieties can beemployed.

As discussed in more detail below, there exist a variety of antigens towhich tolerance may be desired. These may include, but are not limitedto, exogenous antigens that result in an adverse immune response when asubject is exposed to the antigen. In several embodiments, the adverseimmune response could be a result of ingestion of the antigen, e.g.,orally or nasally, or via some other mucosal route. These routes couldbe the case, for example, with food antigens. In some embodiments, theantigen may be purposefully administered to a subject, for example, withthe administration of a therapeutic composition to treat a disease orcondition that the subject is affected by. In still additionalembodiments, the antigen may be produced by the subject, e.g., anautoimmune antigen. For example, in several embodiments, X comprises aforeign transplant antigen against which transplant recipients developan unwanted immune response or a tolerogenic portion thereof. In severalembodiments, X comprises a foreign food, animal, plant or environmentalantigen against which patients develop an unwanted immune response or atolerogenic portion thereof. In several embodiments, X comprises aforeign therapeutic agent against which patients develop an unwantedimmune response or a tolerogenic portion thereof. In severalembodiments, X comprises a synthetic self-antigen against the endogenousversion of which patients develop an unwanted immune response or atolerogenic portion thereof.

In further detail to the above, there are provided in severalembodiments compounds where X is a food antigen. In some suchembodiments, X is one or more of conarachin (Ara h 1), allergen II (Arah 2), arachis agglutinin, conglutin (Ara h 6), a-lactalbumin (ALA),lactotransferrin, Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m2), tropomyosin fast isoform, high molecular weight glutenin, lowmolecular weight glutenin, alpha-gliadin, gamma-gliadin, omega-gliadin,hordein, seclain, and avenin. Fragment of any of these antigens and/ormimotopes of any of these antigens are also used, in severalembodiments. In several embodiments, X is selected from the groupconsisting of gluten, high molecular weight glutenin, low molecularweight glutenin, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein,seclain, and avenin and fragments thereof. In several embodiments, X isselected from the group consisting of gluten, high molecular weightglutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin,and omega-gliadin and fragments thereof. In several embodiments, X isgluten or fragment thereof. In several embodiments, X is gliadin orfragment thereof.

In several embodiments, there are provided compounds where X is atherapeutic agent. In several embodiments, X is selected from the groupconsisting of Factor VII, Factor IX, asparaginase, and uricase andfragments thereof. In several embodiments, X is a therapeutic agentselected from the group consisting of Factor VII and Factor IX andfragments thereof. In several embodiments, X is a therapeutic agentselected from the group consisting of Factor VIII or fragment thereof.In several embodiments, when X is a therapeutic agent, the compound canbe used in the treatment, prevention, reduction, or otherwiseamelioration of an immune response developed against a therapeutic agentfor hemophilia. As discussed herein, mimotopes of any antigenic portionof the antigens above can be used in several embodiments.

In several embodiments, X comprises asparaginase or a fragment thereof.In several embodiments, X comprises uricase or a fragment thereof. Inseveral such embodiments, the compound can be used in the treatment,prevention, reduction, or otherwise amelioration of an immune responsedeveloped against an anti-neoplastic agent. As discussed herein,mimotopes of any antigenic portion of the antigens above can be used inseveral embodiments.

In several embodiments, X is associated with an autoimmune disease. Forexample, in several embodiments, the associated autoimmune disease isone or more of Type I diabetes, multiple sclerosis, rheumatoidarthritis, vitiligo, uveitis, pemphis vulgaris and neuromyelitis optica.

In several embodiments, the autoimmune disease is Type I diabetes and Xcomprises insulin or a fragment thereof. In several embodiments, theautoimmune disease is Type I diabetes and X comprises proinsulin or afragment thereof. In several embodiments, the autoimmune disease is TypeI diabetes and X comprises preproinsulin or a fragment thereof. Asdiscussed herein, mimotopes of any antigenic portion of the antigensabove can be used in several embodiments. In several embodiments,combinations of these antigens can be incorporated into the tolerogeniccompound which may aid in reducing immune responses to self-antigens atmultiple points along the insulin pathway.

In several embodiments, the autoimmune disease is multiple sclerosis andX comprises myelin basic protein or a fragment thereof. In severalembodiments, the autoimmune disease is multiple sclerosis and Xcomprises myelin oligodendrocyte glycoprotein or a fragment thereof. Inseveral embodiments, the autoimmune disease is multiple sclerosis and Xcomprises myelin proteolipid protein or a fragment thereof. As discussedherein, mimotopes of any antigenic portion of the antigens above can beused in several embodiments. In several embodiments, combinations ofthese antigens can be incorporated into the tolerogenic compound whichmay aid in reducing immune responses to self-antigens at multiple pointsalong the enzymatic pathways that control myelination or myelin repair.

In several embodiments, the autoimmune disease is rheumatoid arthritisand X is selected from the group consisting of fibrinogen, vimentin,collagen type II, alpha enolase and fragments thereof.

In several embodiments, the autoimmune disease is vitiligo and X isselected from the group consisting of Pmel17, tyrosinase and fragmentsthereof.

In several embodiments, the autoimmune disease is uveitis and X isselected from the group consisting of retinal arrestin andinterphotoreceptor retinoid-binding protein (IRBP) and fragmentsthereof.

In several embodiments, the autoimmune disease is pemphigus vulgaris andX is selected from the group consisting of desmoglein 3, 1 and 4,pemphaxin, desmocollins, plakoglobin, perplakin, desmoplakins,acetylcholine receptor and fragments thereof.

In several embodiments, the autoimmune disease is neuromyelitis opticaand X is aquaporin-4 or a fragment thereof.

As discussed herein, mimotopes of any antigenic portion of theself-antigens above (or otherwise disclosed herein) can be used inseveral embodiments.

Also provided for in several embodiments is the use of the compoundsdisclosed above (or otherwise disclosed herein) for use in inducingtolerance to X.

There are also provided for in several embodiments hereinpharmaceutically acceptable compositions comprising a compound disclosedabove (or otherwise disclosed herein). There is also provided for theuse of such compositions in inducing tolerance to X. In severalembodiments, the pharmaceutically acceptable composition consists of, orconsists essentially of a compound wherein X is a food antigen,therapeutic agent, a self antigen, or fragment thereof, a linker Y, anda liver targeting moiety Z selected from glucose, galactose,glucosamine, galactosamine, N-acetylglucosamine, andN-acetylgalactosamine.

Also provided for herein are methods of inducing tolerance to an antigento which a subject is capable of developing an unwanted immune response,comprising administering a compounds disclosed above (or otherwisedisclosed herein). In several embodiments, the compound is administeredprior to the subject being exposed to the antigen. However, in severalembodiments, the compound is administered after the subject has beenexposed to the antigen. In several embodiments, the administrationcomprises at least one intravenous administration of the compound (e.g.,a bolus dose followed by a series of optional maintenance doses).

In several embodiments, there is provided for the use of compoundsdisclosed above (or otherwise disclosed herein) in the preparation of amedicament for inducing tolerance to an antigen to which a subjectdevelops an unwanted immune response or a tolerogenic portion thereof.

In several embodiments disclosed herein, there are provided compositionsfor inducing immune tolerance in a subject and methods and uses of thecompositions for achieving the same. In several embodiments, immunetolerance is desired because a subject develops an unwanted immuneresponse to an antigen. Depending on the embodiment, the antigen may beone or more of a variety of antigens, for example a foreign antigen suchas a food antigen that is ingested, or an antigenic portion of atherapeutic drug given to a subject. In additional embodiments, theantigen may be a self-antigen that the subject's immune system fails torecognize (or only recognizes as self to a limited degree) and thereforemounts an immune response against, leading to autoimmune disorders.

In several embodiments, there is provided a composition comprisingFormula 1:X

Y−Z]_(m)   Formula 1wherein m is an integer from about 1 to 10, X comprises a food antigen,a therapeutic agent, a self-antigen, a fragment of any of such antigens,or a mimotope of any of such antigens, Y is of a linker moiety havingthe following formula:

wherein: the left bracket “(” indicates a bond to X, the right or bottombracket and “)” indicates the bond between Y and Z, n is an integer fromabout 70 to 85, where present p is an integer from about 85 to 95, wherepresent q is an integer from about 1 to 10, where present R⁸ is CH₂ orCH₂—CH₂—C(CH₃)(CN); and where present R⁹ is a direct bond orCH₂—CH₂—NHC(O); and Z comprises a liver-targeting moiety comprisingglucose or galactose. In several embodiments, m is between 1 and 3, n is79, p is 90, and q is 4. In several embodiments, X is selected from thegroup consisting of insulin, proinsulin, preproinsulin, gluten, gliadin,myelin basic protein, myelin oligodendrocyte glycoprotein andproteolipid protein, Factor VIII, Factor IX, asparaginase, uricase andfragments of any of the preceding. In several embodiments, thecomposition comprises, consists of, or consists essentially of theantigen X, the linker Y and the liver targeting moiety Z.

In several embodiments, there is provided a compound comprising Formula1:X

Y−Z]_(m)   Formula 1wherein m is an integer from about 1 to 10, X is selected from the groupconsisting of insulin, proinsulin, preproinsulin, gluten, gliadin,myelin basic protein, myelin oligodendrocyte glycoprotein andproteolipid protein, Factor VIII, Factor IX, asparaginase, uricase andfragments of any of the preceding, Y is of a linker moiety having thefollowing formula:

wherein, the left bracket “(” indicates a bond to X, the right or bottombracket and “)” indicates the bond between Y and Z, n is an integer fromabout 70 to 85, where present p is an integer from about 85 to 95, wherepresent q is an integer from about 1 to 10, where present R⁸ is CH₂ orCH₂—CH₂—C(CH₃)(CN); and where present R⁹ is a direct bond orCH₂—CH₂—NHC(O), and Z comprises a liver-targeting moiety comprising asugar moiety. In several embodiments, m is between 1 and 3, n is 79, pis 90, and q is 4. In several embodiments, Z is selected from the groupconsisting of glucose, glucosamine, galactose, galactosamine,N-acetylgalactosamine and N-acetylglucosamine.

In several embodiments, 2,5-dioxopyrrolidin-1-yl propylcarbonate-linkers and/or 2-(ethyldisulfanyl)ethyl ethylcarbamate-linkerscan be used.

In several embodiments, there is provided a composition comprising acompound of Formula 1:X

Y−Z]_(m)   Formula 1wherein:

m is an integer from about 1 to 10;

X comprises an antigen to which patients develop an unwanted immuneresponse, wherein the antigen is a food antigen, a therapeutic agent, aself-antigen, or a fragment of any of such antigens;

Y is of a linker moiety having a formula selected from the groupconsisting of:

wherein the left bracket “(” indicates a bond to X, where present theright “)” indicates a bond to Z, where present the bottom “)” indicatesa bond to Z, where present n is an integer from about 1 to about 80,where present q is an integer from about 1 to about 4, where present pis an integer from about 1 to about 90, where present R₈ is CH₂ orCH₂—CH₂—C(CH₃)(CN), and Z comprises one or more liver-targeting moietiesthat specifically target liver cells expressing asialoglycoproteinreceptors.

In several embodiments of the composition, m is 1 to 4, Y is of a linkermoiety having a formula of:

and Z comprises a liver-targeting moiety comprising one or more ofgalactose, galactosamine, or N-acetyl galactosamine.

In several embodiments, m is resolved to an integer from 1 to 4, Y is ofa linker moiety having a formula of:

and Z comprises a liver-targeting moiety comprising one or more ofglucose, glucosamine, or N-acetyl glucosamine.

In several embodiments, there is provided compositions of Formula 1 (X

Y—Z]_(m)), where m is an integer from about 1 to 100, X comprises anantigen against which a patient develops an unwanted immune response, ora tolerogenic portion thereof or X comprises an antibody, antibodyfragment or ligand that specifically binds a circulating protein orpeptide or antibody, which circulating protein or peptide or antibody iscausatively involved in transplant rejection, immune response against atherapeutic agent, autoimmune disease, hypersensitivity and/or allergy,Y comprises a linker moiety, and Z comprises a liver-targeting moiety.In several embodiments, Z comprises galactose, galactosamine,N-acetylgalactosamine, glucose, glucosamine or N-acetylglucosamine.

In several embodiments, Y is selected from N-hydroxysuccinamidyllinkers, malaemide linkers, vinylsulfone linkers, pyridyldi-thiol-poly(ethylene glycol) linkers, pyridyl di-thiol linkers,n-nitrophenyl carbonate linkers, NHS-ester linkers, and nitrophenoxypoly(ethylene glycol)ester linkers. In some embodiments, Y comprises anantibody, antibody fragment, peptide or other ligand that specificallybinds X, a disulfanyl ethyl ester, a structure represented by one ofFormulae Ya to Yp:

or Y has a portion represented by Formula Y′-CMP:

In such embodiments, the left bracket “(” indicates the bond between Xand Y, the right or bottom bracket and “)” indicates the bond between Yand Z, n is an integer from about 1 to 100, q is an integer from about 1to 44, R⁸ is CH₂ or CH₂—CH₂—C(CH₃)(CN), Y′ represents the remainingportion of Y; and W represents a polymer of the same W¹ group, or W is acopolymer or a random copolymer of the same or different W¹ and W²groups, where:

and where p is an integer from 2 to about 150, R⁹ is a direct bond,—CH₂—CH₂—NH—C(O)— or —CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—, t is an integerfrom 1 to 5; and R¹⁰ is an aliphatic group, an alcohol or an aliphaticalcohol. In one such embodiment, m is 1 to 3, Y is represented byFormula Ym, wherein R⁸ is —CH₂—CH₂—C(CH₃)(CN)—, and W is represented bya block copolymer of W¹ and W² where R⁹ is—CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—, t is 1, and R¹⁰ is 2-hydroxypropyl;and Z comprises a liver-targeting moiety comprising one or more ofgalactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine,N-acetylglucosamine. In several embodiments, Z is the β-anomer of thecorresponding sugar.

In several additional embodiments compositions are provided for inducingtolerance to an antigen to which a subject develops an unwanted immuneresponse, the compositions comprising a compound of Formula 1 (Formula 1(X

Y—Z]_(m)), where m is an integer from about 1 to 10, X comprises anantigen to which patients develop an unwanted immune response, whereinthe antigen is a food antigen, a therapeutic agent, a self-antigen, or afragment of any of such antigens, Y is of a linker moiety having aformula selected from the group consisting of:

wherein the left bracket “(” indicates a bond to X, the right or bottombracket and “)” indicates the bond between Y and Z, n is an integer fromabout 1 to 100, where present p is an integer from about 2 to 150, wherepresent q is an integer from about 1 to 44, where present R⁸ is CH₂ orCH₂—CH₂—C(CH₃)(CN), and where present R⁹ is a direct bond orCH₂—CH₂—NHC(O), and Z comprises galactose, galactosamine, orN-acetylgalactosamine.

In several embodiments of such compositions, m is 1 to 3, Y is of alinker moiety having a formula of:

wherein CH₂—CH₂—NHC(O); and Z comprises a liver-targeting moietycomprising one or more of galactose, galactosamine, orN-acetylgalactosamine. In several embodiments, Z is the β-anomer of theselected moiety.

As discussed above, in several embodiments, X is a self-antigen and theunwanted immune response is an autoimmune response.

A variety of self-antigens is disclosed herein, but in severalparticular embodiments, X is myelin oligodendrocyte glycoprotein ormyelin proteolipid protein. In such embodiments, the unwanted immuneresponse experienced by the subject is associated with multiplesclerosis. In additional embodiments, X is insulin, proinsulin, orpreproinsulin and wherein the unwanted immune response is associatedwith diabetes mellitus. It shall be appreciated that being associatedwith multiple sclerosis, diabetes mellitus or other auto-immune diseaseneed not necessarily require a formal diagnosis of such auto-immunecondition, but rather can be associated with common symptoms orcharacteristics of a particular auto-immune disorder.

In additional embodiments, as discussed herein, an unwanted immuneresponse can be raised against a therapeutic agent, such as a proteindrug or drug derived from non-human and/or non-mammalian species. Forexample, in several embodiments, X is a therapeutic agent, such asFactor VIII, Factor IX, or other hemostasis-inducing agent. In suchembodiments, the unwanted immune response is against the agent and theassociated disease is hemophilia, which fails to improve (in the absenceof the composition) because of the autoimmune response. However, uponadministration of the composition, the hemophilia can improve becausethe composition aids in inducing tolerance to the agent, reducing theresponse to agent, and allowing reduced symptoms of hemophilia. In stilladditional embodiments, X is a therapeutic agent such as asparaginaseand uricase. As discussed above, an unwanted immune response can resultfrom administration of such agents, as they are derived from non-humansources. The ability of the compositions disclosed herein to inducetolerance to these agents allows these agents to continue to be used bya subject in need of therapy, while the side effects from an immunereaction are reduced, lessened, eliminated or otherwise ameliorated.

In several embodiments, X is a food antigen. Many food antigens areknown to cause allergies upon ingestion, however, in severalembodiments, X is selected from the group consisting of conarachin (Arah 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6),a-lactalbumin (ALA), lactotransferrin, Pen a 1 allergen (Pen a 1),allergen Pen m 2 (Pen m 2), tropomyosin fast isoform, high molecularweight glutenin, low molecular weight glutenin, alpha-gliadin,gamma-gliadin, omega-gliadin, hordein, seclain, and avenin. In severalembodiments, treatment with the compositions disclosed herein where X isa food antigen allows the subject to have a significantly reduced immuneresponse to the antigen, e.g., many peanut allergies are so severe thatexposure to peanut dust or oil can cause anaphylaxis. In someembodiments, treatment reduces and/or eliminates responses to suchincidental exposure to the antigen. In additional embodiments, treatmentallows the subject to ingest the food from which the antigen is derivedwith limited or no adverse immune response.

In several embodiments, administration of the composition to the subjectresults in a greater degree of proliferation of antigen-specific T cellsas compared to proliferation of antigen-specific T cells resulting fromadministration of the antigen alone. In such embodiments, theproliferation of antigen-specific T cells indicates that delivery of theantigen (via the composition) to the molecular processing machinery thatprocesses antigens as self/non-self is enhanced versus administration ofthe antigen alone. In other words, in such embodiments the targeteddelivery is effective. In still additional embodiments, administrationof the compositions disclosed herein results in a greater expression ofexhaustion markers or markers of apoptosis on antigen-specific T cellsas compared to expression of exhaustion markers or markers of apoptosison antigen-specific T cells resulting from administration of the antigenalone. This result in indicative of specific reduction in activity of Tcells directed against the antigen of interest and/or deletion of Tcells directed against the antigen of interest. In several embodiments,these molecular hallmarks of induction of tolerance are the precursor ofthe reduction or amelioration of immune response symptoms that thesubject would have previously experienced when exposed to the antigen.

In several embodiments, Z comprises a liver-targeting moiety that is acarbohydrate. In several embodiments, the carbohydrate is a short-chaincarbohydrate. In several embodiments, Z is a sugar. In severalembodiments, Z is galactose, galactosamine, N-acetylgalactosamine,glucose, glucosamine, or N-acetylglucosamine. In several embodiments,the induction of immune tolerance is greater when a glucose,glucosamine, or N-acetylglucosamine is used for Z. In still additionalembodiments, enhancements in induction of immune tolerance can beachieved when the liver targeting moiety is a sugar and the sugar is inthe 6-anomer configuration. In several embodiments, Z is galactose,galactosamine, N-acetylgalactosamine, glucose, glucosamine, orN-acetylglucosamine and conjugated at its C1, C2 or C6 to Y.

Also provided herein are methods of inducing tolerance to antigenswhich, when administered alone (e.g., without the presently disclosedcompositions) would result in an adverse immune response. Such methods,depending on the embodiments, involved the administration either before,or after, exposure to the antigen. In several embodiments,administration prior to exposure serves a prophylactic effect, which inseveral embodiments essentially avoids or significantly reduces in theimmune response. Administration of the compositions can be via a varietyof methods, including, but not limited to intravenous, intramuscular,oral, transdermal, or other infusion route. Administration can be daily,weekly, multiple times per day, or on an as needed basis (e.g., prior toan anticipated exposure).

Also provided for herein are uses of the compositions disclosed hereinfor the treatment of unwanted immune responses after exposure to anantigen. As discussed herein, such use can be for prophylactic effectsand/or for reducing symptoms from prior exposure to antigens (or prioradverse immune effects, such as those in the auto-immune setting). Forexample, provided herein are uses of compositions according to Formula 1for the treatment of unwanted side effects due to exposure to atherapeutic antigen, exposure to a food antigen, or an adverse effectfrom an immune response against a self-antigen. The compositionsdisclosed herein are suitable for administration to a subject inconjunction with such use, for example by oral, IV, IM, or othersuitable route. Uses of the compositions disclosed herein, in severalembodiments, unexpectedly result in the reduction, elimination oramelioration of adverse immune responses to antigens of interest.

Additional compositions and methods of using them are provided herein.For example, in several embodiments, there is provided apharmaceutically acceptable composition for inducing tolerance to atherapeutic protein in a subject having an deficiency in production of afunctional analogous native protein, comprising a compound of Formula 1(X

Y—Z]_(m)), where m is an integer from about 1 to 10, X comprises anantigenic protein or protein fragment, Y is of a linker moiety having aformula selected from the group consisting of Formula Ya, Formula Yc,Formula Ym, Formula Yn, wherein, the left bracket “(” indicates a bondto X, the right or bottom bracket and “)” indicates the bond between Yand Z, n is an integer from about 1 to 100, where present p is aninteger from about 2 to 150, where present q is an integer from about 1to 44, where present R⁸ is —CH₂— or —CH₂—CH₂—C(CH₃)(CN)—, where presentR⁹ is a direct bond or —CH₂—CH₂—NH—C(O)—, and Z comprises galactose,galactosamine, or N-acetylgalactosamine.

In several embodiments of the composition, m is 1 to 3, Y is of a linkermoiety having a formula of:

wherein CH₂—CH₂—NH—C(O)—, and Z comprises a liver-targeting moietycomprising one or more of glucose, glucosamine, N-acetylglucosamine,galactose, galactosamine, or N-acetylgalactosamine. In severalembodiments, the galactose, galactosamine, or N-acetylgalactosamine arethe β-anomers. In several embodiments, combinations of galactose,galactosamine, N-acetylgalactosamine, glucose, glucosamine, orN-acetylglucosamine are used.

Also provided for herein is a pharmaceutically acceptable compositionfor inducing tolerance to a therapeutic protein in a subject having andeficiency in production of a functional analogous native protein,comprising a compound of Formula 1 (X

Y—Z]_(m)), where m is an integer from about 1 to 10, X comprises aantigenic protein or protein fragment, Y is of a linker moiety having aformula selected from the group consisting of Formula Ya, Formula Yc,Formula Ym, or Formula Ym, wherein the left bracket “(” indicates a bondto X, where present the right “)” indicates a bond to Z, where presentthe bottom “)” indicates a bond to Z, where present n is an integer fromabout 1 to about 80, where present q is an integer from about 1 to about4, where present p is an integer from about 1 to about 90, where presentR⁸ is —CH₂— or —CH₂—CH₂—C(CH₃)(CN)—, and where present W represents apolymer of the Formula W¹ or W² group or W is a copolymer of Formula W¹or W² where:

where R⁹ is a direct bond, —CH₂—CH₂—NH—C(O)— or—CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—, t is an integer from 1 to 5, R¹⁰ isan aliphatic group, an alcohol or an aliphatic alcohol; and Z comprisesglucose, glucosamine, N-acetylglucosamine, galactose, galactosamine, orN-acetylgalactosamine. In several embodiments, the galactose,galactosamine, or N-acetylgalactosamine are the β-anomers. In severalembodiments, combinations of galactose, galactosamine,N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine areused. In several embodiments of the composition, m is 1 to 3, Y isrepresented by Formula Ym, wherein R⁸ is CH₂—CH₂—C(CH₃)(CN), and W isrepresented by a block copolymer of W¹ and W² where R⁹ is—CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—, t is 1, and R¹⁹ is 2-hydroxypropyl;and Z comprises a liver-targeting moiety comprising one or more ofglucose, glucosamine, N-acetylglucosamine, galactose, galactosamine, orN-acetylgalactosamine. In several embodiments, the galactose,galactosamine, or N-acetylgalactosamine are the β-anomers. In severalembodiments, combinations of galactose, galactosamine,N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine areused.

In several embodiments, X comprises an antigenic region of myelin basicprotein, myelin oligodendrocyte glycoprotein, or myelin proteolipidprotein. In additional embodiments, X comprises an antigenic region ofFactor VIII, Factor IX, insulin, uricase, PAL, or asparaginase. Inadditional embodiments, X comprises a foreign antigen such as conarachin(Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h6), a-lactalbumin (ALA), lactotransferrin, Pen a 1 allergen (Pen a 1),allergen Pen m 2 (Pen m 2), tropomyosin fast isoform, high molecularweight glutenin, low molecular weight glutenin, alpha-gliadin,gamma-gliadin, omega-gliadin, hordein, seclain, and avenin.

Additionally provided for herein are compositions comprising a compoundof Formula 1 (X

Y—Z]m), where m is an integer from about 1 to 100, X comprises anantigen against which a patient develops an unwanted immune response, ora tolerogenic portion thereof, or X comprises an antibody, antibodyfragment or ligand that specifically binds a circulating protein orpeptide or antibody, which circulating protein or peptide or antibody iscausatively involved in transplant rejection, immune response against atherapeutic agent, autoimmune disease, hypersensitivity and/or allergy,Y comprises a linker moiety, and Z comprises a liver-targeting moiety.

In several embodiments, Z galactose, galactosamine,N-acetylgalactosamine, glucose, glucosamine or N-acetylglucosamine.Combinations of galactose, galactosamine, N-acetylgalactosamine,glucose, glucosamine or N-acetylglucosamine may also be used, in severalembodiments. Further, in several embodiments, the galactose,galactosamine, N-acetylgalactosamine, glucose, glucosamine orN-acetylglucosamine are optionally the β, anomer. In severalembodiments, Z is conjugated at its C1, C2 or C6 to Y.

In several embodiments, Y is selected from N-hydroxysuccinamidyllinkers, malaemide linkers, vinylsulfone linkers, pyridyldi-thiol-poly(ethylene glycol) linkers, pyridyl di-thiol linkers,n-nitrophenyl carbonate linkers, NHS-ester linkers, and nitrophenoxypoly(ethylene glycol)ester linkers. In several embodiments, Y comprisesan antibody, antibody fragment, peptide or other ligand thatspecifically binds X, a disulfanyl ethyl ester, a structure representedby one of Formulae Ya to Yp, or Y has a portion represented by FormulaY′-CMP:

where the left bracket “(” indicates the bond between X and Y, the rightor bottom bracket and “)” indicates the bond between Y and Z, n is aninteger from about 1 to 100, q is an integer from about 1 to 44, R⁸ isCH₂ or CH₂—CH₂—C(CH₃)(CN), Y′ represents the remaining portion of Y, andW represents a polymer of the same W¹ group, or W is a copolymer or arandom copolymer of the same or different W¹ and W² groups, where:

where p is an integer from 2 to about 150, R⁹ is a direct bond,CH₂—CH₂—NHC(O) or —CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—, t is an integerfrom 1 to 5; and R¹⁹ is an aliphatic group, an alcohol or an aliphaticalcohol.

In some such embodiments, n is about 40 to 80, p is about 10 to 100, qis about 3 to 20, R⁸ is CH₂—CH₂—C(CH₃)(CN), when R9 is CH₂—CH₂—NHC(O), Zis glucose, galactose, N-acetylgalactosamine or N-acetylglucosamineconjugated at its C1, and when W is a copolymer, R10 is 2-hydroxypropyl.In some embodiments, Y comprises Formula Ya, Formula Yb, Formula Yc,Formula Yf, Formula Yg, Formula Yh, Formula Yi, Formula Yk, Formula Ymor Formula Yn. In some embodiments, Y comprises Formula Ya, Formula Yb,Formula Yc, Formula Ym or Formula Yn. In still additional embodiments, Ycomprises Formula Ya, Formula Yb, Formula Yc, Formula Ym or Formula Yn.

In several embodiments, X comprises a foreign transplant antigen againstwhich transplant recipients develop an unwanted immune response, aforeign food, animal, plant or environmental antigen against whichpatients develop an unwanted immune response, a foreign therapeuticagent against which patients develop an unwanted immune response, or asynthetic self-antigen against the endogenous version of which patientsdevelop an unwanted immune response, or a tolerogenic portion thereof.Specific examples of various antigens are disclosed herein.

Also provided for herein is are methods of treatment for an unwantedimmune response against an antigen by administering to a mammal in needof such treatment an effective amount of a composition comprising acompound of Formula 1 (X

Y—Z]m), where m is an integer from about 1 to 100, X comprises anantigen against which a patient develops an unwanted immune response, ora tolerogenic portion thereof or X comprises an antibody, antibodyfragment or ligand that specifically binds a circulating protein orpeptide or antibody, which circulating protein or peptide or antibody iscausatively involved in transplant rejection, immune response against atherapeutic agent, autoimmune disease, hypersensitivity and/or allergy,Y comprises a linker moiety, and Z comprises a glucosylatedliver-targeting moiety.

In several such embodiments, X comprises an antigen against which apatient develops an unwanted immune response, or a tolerogenic portionthereof, and Y comprises, an antibody, antibody fragment, peptide orother ligand that specifically binds X, a disulfanyl ethyl ester, astructure represented by one of Formulae Ya to Yp or Y has a portionrepresented by Formula Y′-CMP where, the left bracket “(” indicates thebond between X and Y, the right or bottom bracket and “)” indicates thebond between Y and Z, n is an integer from about 1 to 100, q is aninteger from about 1 to 44, R⁸ is CH₂ or CH₂—CH₂—C(CH₃)(CN), Y′represents the remaining portion of Y, and W represents a polymer of thesame W¹ group, or W is a copolymer or a random copolymer of the same ordifferent W¹ and W² groups, where:

where p is an integer from 2 to about 150, R⁹ is a direct bond,CH₂—CH₂—NHC(O) or —CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—, t is an integerfrom 1 to 5, and R¹⁹ is an aliphatic group, an alcohol or an aliphaticalcohol. In several such treatment method embodiments, X comprises theantibody, antibody fragment or ligand, and the composition isadministered for clearance of a circulating protein or peptide orantibody that specifically binds to X, which circulating protein orpeptide or antibody is causatively involved in transplant rejection,immune response against a therapeutic agent, autoimmune disease,hypersensitivity and/or allergy.

In still additional embodiments, X comprises the antibody, antibodyfragment or ligand, and the composition is administered in an amounteffective to reduce a concentration of the antibodies that arecausatively involved in transplant rejection, immune response against atherapeutic agent, autoimmune disease, hypersensitivity and/or allergyin blood of the patient by at least 50% w/w, as measured at a timebetween about 12 to about 48 hours after the administration.

In several such treatment embodiments, compositions are administered fortolerization of the patient with respect to antigen moiety X.

In several embodiments X comprises a foreign transplant antigen againstwhich transplant recipients develop an unwanted immune response, aforeign food, animal, plant or environmental antigen against whichpatients develop an unwanted immune response, a foreign therapeuticagent against which patients develop an unwanted immune response, or asynthetic self-antigen against the endogenous version of which patientsdevelop an unwanted immune response, or a tolerogenic portion thereof.

Several embodiments disclosed herein provide a composition comprising acompound of Formula 1:X

Y−Z]_(m)   Formula 1where:

-   -   m is an integer from about 1 to 100;    -   X comprises an antigen against which a patient develops an        unwanted immune response, or a tolerogenic portion thereof; or    -   X comprises an antibody, antibody fragment or ligand that        specifically binds a circulating protein or peptide or antibody,        which circulating protein or peptide or antibody is causatively        involved in transplant rejection, immune response against a        therapeutic agent, autoimmune disease, hypersensitivity and/or        allergy;    -   Y comprises a linker moiety; and    -   Z comprises a liver-targeting moiety.

Z can also comprise galactose, galactosamine, N-acetylgalactosamine,glucose, glucosamine or N-acetylglucosamine, for example, conjugated atits C1, C2 or C6 to Y. N-acetylglucosamine and glucose bind to differentlectin receptors as do N-acetylgalactosamine and galactose. In theexamples below the experimental data (and the full disclosure of thisapplication) indicate that the selection of Z as N-acetylglucosamineleads to elevated levels of regulatory T cell responses compared tothose achieved with N-acetylgalactosamine. In several embodiments, thisresults in unexpectedly enhanced induction of immune tolerance and/orclearance of antigens from the blood of a subject.

Y can be selected from N-hydroxysuccinamidyl linkers, malaemide linkers,vinylsulfone linkers, pyridyl di-thiol-poly(ethylene glycol) linkers,pyridyl di-thiol linkers, n-nitrophenyl carbonate linkers, NHS-esterlinkers, and nitrophenoxy poly(ethylene glycol)ester linkers.

Y can also comprise: an antibody, antibody fragment, peptide or otherligand that specifically binds X; a disulfanyl ethyl ester; a structurerepresented by one of Formulae Ya to Yp:

or Y has a portion represented by Formula Y′-CMP:

where:

-   -   the left bracket “(” indicates the bond between X and Y;    -   the right or bottom bracket and “)” indicates the bond between Y        and Z;    -   n is an integer from about 1 to 100;    -   q is an integer from about 1 to 44;    -   R⁸ is CH₂ or CH₂—CH₂—C(CH₃)(CN);    -   Y′ represents the remaining portion of Y (e.g., HS-PEG); and    -   W represents a polymer of the same W¹ group, or W is a copolymer        (preferably a random copolymer) of the same or different W¹ and        W² groups, where:

-   -   where:        -   p is an integer from 2 to about 150;        -   R⁹ is a direct bond, CH₂—CH₂—NHC(O) (i.e., an ethylacetamido            group or “EtAcN”) or —CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—            (i.e., a pegylated ethylacetamido group or “Et-PEG_(t)-AcN”)        -   t is an integer from 1 to 5, (particularly 1 to 3, and more            particularly 1 or 2); and        -   R¹⁰ is an aliphatic group, an alcohol or an aliphatic            alcohol. In some embodiments, R¹⁹ is a C_(f)alkyl or            C_(f)alkylOH_(g) where f is independently an integer between            0 and 10 and g is independently an integer between 0 and 10.            In some embodiments, R¹⁹ is 2-hydroxypropyl.

In several embodiments, particular linkers are preferred. For example,in several embodiments, linkers according to Ym yield unexpectedlyeffective tolerance endpoints. In additional embodiments, linkersaccording to formula Yn yield unexpectedly effective toleranceendpoints. In still additional embodiments, formulations ofF1m′-OVA-m₁₋₃-n₇₉-p₉₀-q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀achieve particularly effective tolerance-associated endpoints. Inseveral embodiments, combinations of these linkers lead to synergisticresults and still further unexpected increases in immune toleranceinduction.

In another aspect of the above, n is about 40 to 80, p is about 10 to100, q is about 3 to 20, R⁸ is CH₂—CH₂—C(CH₃)(CN); and when R⁹ isCH₂—CH₂—NHC(O), Z is galactose or N-acetylgalactosamine conjugated atits C1.

In still another aspect of the above, Y comprises Formula Ya, FormulaYb, Formula Yh, Formula Yi, Formula Yk, Formula Ym or Formula Yn,particularly Formula Ya, Formula Yb, Formula Ym or Formula Yn.

X can further comprise: a foreign transplant antigen against whichtransplant recipients develop an unwanted immune response; a foreignfood, animal, plant or environmental antigen against which patientsdevelop an unwanted immune response; a foreign therapeutic agent againstwhich patients develop an unwanted immune response; or a syntheticself-antigen against the endogenous version of which patients develop anunwanted immune response, or a tolerogenic portion thereof.

The disclosure also pertains to a method of treatment for an unwantedimmune response against an antigen by administering to a mammal in needof such treatment an effective amount of a composition comprising acompound of Formula 1 as disclosed herein. In some such methods thecomposition can be administered for clearance of a circulating proteinor peptide or antibody that specifically binds to antigen moiety X,which circulating protein or peptide or antibody is causatively involvedin transplant rejection, immune response against a therapeutic agent,autoimmune disease, hypersensitivity and/or allergy. The composition canbe administered in an amount effective to reduce a concentration of theantibodies that are causatively involved in transplant rejection, immuneresponse against a therapeutic agent, autoimmune disease,hypersensitivity and/or allergy in blood of the patient by at least 50%w/w, as measured at a time between about 12 to about 48 hours after theadministration. The composition can administered for tolerization of apatient with respect to antigen moiety X.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a series of graphs showing differential cellular uptakeof galactose conjugates. FIG. 1A depicts that F1aA-PE-m₄-n₈₀ (Gal-PE)preferentially targets PE to sinusoidal endothelial cells (LSECs) of theliver. FIG. 1B depicts that F1aA-PE-m₄-n₈₀ (Gal-PE) preferentiallytargets PE to Kupffer cells (KC) of the liver. FIG. 1C depicts thatF1aA-PE-m₄-n₈₀ (Gal-PE) preferentially targets PE to hepatocytes. FIG.1D depicts that F1aA-PE-m₄-n₈₀ (Gal-PE) preferentially targets PE toother antigen presenting cells (APCs) of the liver. *=P<0.05.

FIG. 2 is a graph showing proliferation of OT-1 CD8+ T cells in micetreated with F1aA-OVA-m₄-n₈₀ (Gal-OVA), OVA or saline (i.e. nave), withgreatest proliferation seen in the Gal-OVA treated group.

FIGS. 3A-3B are a series of graphs depicting data related to markerexpression on T cells. FIG. 3A shows the percentage of OT-1 CD8⁺ T cellsexpressing PD-1 (“PD1+”) in generations of proliferating T cells treatedwith saline, OVA or F1aA-OVA-m₄-n₈₀ (GAL-OVA), with greatest level ofPD-1 in the gal-OVA-treated group. FIG. 3B shows the percentage of OT-1CD8⁺ T cells expressing phosphatidylserine (stained as “Annexin V+”) ingenerations of proliferating T cells treated with saline, OVA orF1aA-OVA-m₄-n₈₀ (GAL-OVA), with greatest level of Annexin-V+ cells inthe gal-OVA-treated group.

FIG. 4 is a graph showing that galactose conjugation [F1aA-OVA-m₄-n₈₀(Gal-OVA)] decreases the immunogenicity of OVA as determined byOVA-specific antibody titers (shown in Ab titers log⁻¹).

FIG. 5 shows that administration of F1aA-OVA-m₄-n₈₀ (Gal-OVA) inrepeated doses over time is able to deplete OVA-specific antibodies fromthe serum of mice.

FIGS. 6A-6F depict data related to the mitigation of the OVA-specificimmune response. FIG. 6A shows the immune response in mice challengedwith OVA and LPS. FIG. 6B shows the immune response in mice treated withOVA, while FIG. 6C shows the immune response in nave mice.

FIGS. 6D and 6E (respectively) show that F1aA-OVA-m₄-n₈₀ (mGal-OVA; 6D)and F1b-OVA-m₁-n₄₄-p₃₄ (pGal-OVA; 6E) are able to mitigate theOVA-specific immune response in draining lymph nodes after intradermalchallenge with OVA and the adjuvant LPS. FIG. 6F is from a parentapplication and does not form a part of the present disclosure.

FIGS. 7A-7B shows the characterization of F1aA-OVA-m₄-n₈₀ andF1b-OVA-m₁-n₄₄-p₃₄. FIG. 7A shows size-exclusion HPLC traces ofF1aA-OVA-m₄-n₈₀ (open triangles), F1b-OVA-m₁-n₄₄-p₃₄ (filled circles)and unconjugated OVA (solid line). Shift to the left represents anincrease in molecular weight. FIG. 7B shows polyacrylamide geldemonstrating increased molecular weight after OVA conjugation: (1.)Unconjugated OVA, (2.) F1aA-OVA-m₄-n₈₀ and (3.) F1b-OVA-m₁-n₄₄-p₃₄.

FIGS. 8A-8B depict data related to the reduction in antigen-specificimmune response after administration ofF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀ [labeledOVA-p(Glu-HPMA) and shown as filled circles] orF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ [labeledOVA-p(Gal-HPMA) and shown as filled diamonds]. FIG. 8A depicts flowcytometric detection of OTI CD8+ T-cell populations(CD3e⁺/CD8α⁺/CD45.2⁺) quantified from the draining lymph nodes (inguinaland popliteal) 4 days following antigen challenge in CD45.1⁺ mice.Significant reductions in OT-I CD8+ T-cells were detected followingadministration of OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA). FIG. 8B depictsflow cytometric detection of OTII CD4+ T-cell populations(CD3e⁺/CD4⁺/CD45.2⁺) quantified from the draining lymph nodes (inguinaland popliteal) 4 d following antigen challenge in CD45.1⁺ mice.Significant reductions in OT-II CD4+ T-cells were detected followingadministration of OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) *=P<0.05,**=P<0.01; #=P<0.05, ##=P<0.01 (#'s represent significance as comparedto nave animals).

FIGS. 9A-9B depict data related to the increase in antigen-specificregulatory T-cells in the lymph nodes and spleen of mice after antigenchallenge. FIG. 9A depicts flow cytometric detection of anF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀ [labeledOVA-p(Glu-HPMA) and shown as filled circles] andF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ [labeledOVA-p(Gal-HPMA) and shown as filled diamonds]-induced increase in OTIIT-regulator cells (CD3e+CD4+CD45.2+CD25+FoxP3+) collected from the lymphnodes 4 d following antigen challenge in CD45.1+ mice. FIG. 9B shows thecorresponding analysis from the spleen of mice treated withOVA-p(Glu-HPMA) or OVA-p(Gal-HPMA) as compared to animals treated withOVA or saline (i.e. Challenge) *=P<0.05, **=P<0.01; ***=P<0.001;#=P<0.01, ##=P<0.01; ###=P<0.001 (#'s represent significance as comparedto nave animals).

FIG. 10 depicts flow cytometry data related to a decrease in thepercentage of antigen-specific effector cells (IFNγ+OTI CD8+ T-cells(CD3e+CD8α+CD45.2+IFNγ+) 4 d following antigen challenge in CD45.1+mice. Mice treated withF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀ [labeledOVA-p(Glu-HPMA) and shown as filled circles] orF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ [labeledOVA-p(Gal-HPMA) and shown as filled diamonds] conjugates generatedsignificantly fewer IFNγ+OTI CD8+ T-cells after antigen challenge ascompared to mice treated with OVA or saline (i.e. Challenge) *=P<0.01,**=P<0.01; ##=P<0.01 (#'s represent significance as compared to naveanimals).

FIGS. 11A-11B depict data related to T cell deletion and regulation inan OTII adoptive transfer model, in which OTII cells (CD4⁺ T cells froma CD45.2⁺ mouse) are adoptively transferred into a CD45.1⁺ recipient,which is treated withF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀[“OVA-p(Gal-HPMA)”] orF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀[“OVA-p(Glu-HPMA)”], or OVA not linked to a polymer [“OVA”] to induce Tregulatory responses and prevent subsequent responses tovaccine-mediated antigen challenge. Both 3×10⁵ CFSE-labeled OTI and3×10⁵ CFSE-labeled OTII cells were adoptively transferred to CD45.1⁺mice (n=8 mice per group) on day 0. On days 1, 4 and 7, tolerogenicdoses or control doses were administered. In one regimen, OVA wasprovided at a dose of 2.5 μg at day 1, 2.5 μg at day 4, and 16 μg at day7. In another, OVA was provided at a dose of 7 μg at day 1, 7 μg at day4, and 7 μg at day 7, for the same total dose. Likewise, pGal-OVA andpGlu-OVA were each administered in other groups at the same dosings of2.5 μg at day 1, 2.5 μg at day 4, and 16 μg at day 7 or 7 μg at day 1, 7μg at day 4, and 7 μg at day 7, all doses being on an OVA equivalentdose basis. In a final group, saline was administered on the same days.On day 14, the recipient mice were then challenged with OVA (10 μg)adjuvanted with lipopolysaccharide (50 ng) by intradermal injection.Characterization of the draining lymph nodes was done on day 19, toallow determination as to whether or not deletion actually took placeand whether regulatory T cells were induced from the adoptivelytransferred cells. FIG. 11A shows the number of OTII cells present afterchallenge, and FIG. 11B shows the frequency of FoxP3⁺ CD25⁺ cells(markers of T regulatory cells). * and # indicate p<0.05, ** and ##indicate p<0.01, and ### indicates P<0.001.

FIGS. 12A-12B depicts data related to T cell deletion and regulation inan OTI adoptive transfer model, in which OTI cells (CD8⁺ T cells from aCD45.2⁺ mouse) are adoptively transferred into a CD45.1⁺ recipient,which is treated withF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀[“OVA-p(Gal-HPMA)”] orF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀[“OVA-p(Glu-HPMA)”], or OVA not linked to a polymer [“OVA”] to induce Tregulatory responses and prevent subsequent responses tovaccine-mediated antigen challenge. Both 3×10⁵ CFSE-labeled OTI and3×10⁵ CFSE-labeled OTII cells were adoptively transferred to CD45.1⁺mice (n=8 mice per group) on day 0. On days 1, 4 and 7, tolerogenicdoses or control doses were administered. In one regimen, OVA wasprovided at a dose of 2.5 μg at day 1, 2.5 μg at day 4, and 16 μg at day7. In another, OVA was provided at a dose of 7 μg at day 1, 7 μg at day4, and 7 μg at day 7, for the same total dose. Likewise, pGal-OVA andpGlu-OVA were each administered in other groups at the same dosings of2.5 μg at day 1, 2.5 μg at day 4, and 16 μg at day 7 or 7 μg at day 1, 7μg at day 4, and 7 μg at day 7, all doses being on an OVA equivalentdose basis. In a final group, saline was administered on the same days.On day 14, the recipient mice were then challenged with OVA (10 μg)adjuvanted with lipopolysaccharide (50 ng) by intradermal injection.Characterization of the draining lymph nodes was done on day 19, toallow determination as to whether or not deletion actually took placeand whether T cells were responsive to antigen re-exposure though theircytokine expression. FIG. 12A shows the number of OTI cells presentafter challenge, and FIG. 12B shows the frequency of IFNγ-expressingcells (lack thereof indicating anergy). * and # indicate p<0.05, ** and## indicate p<0.01).

FIG. 13 depicts data related to blood glucose levels. Mice were treatedwith F1m′-P31-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀[labeled P31-p(Glu-HPMA)],F1m′-P31-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ [labeledP31-p(Gal-HPMA) conjugates (or saline). Animals receivingP31-p(Glu-HPMA) or P31-p(Gal-HPMA) maintained normal blood glucoselevels for 42 days, whereas animals treated with P31 or Saline developedrapid hyperglycemia within 5-10 days, demonstrating that conjugatesdisclosed herein protect mice from T-cell induced autoimmune diabetes.

FIG. 14 depicts data related to the generation of spontaneous diabetesin non-obese diabetic (NOD) mice. Mice treated withF1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀ areshown as filled squares. Mice treated withF1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ areshown as filled triangles. Mice treated with saline are shown as filleddiamonds. Treating animals with the compounds of Formula 1 reduced theincidences of diabetes onset in NOD mice as compared to animals treatedwith saline.

FIGS. 15A-15B depicts data related to biodistribution of the modelantigen OVA tethered to the synthetic glycopolymers, showing uptake inthe liver while limiting uptake in the spleen. A. Fluorescent signal ofperfused livers taken from animals treated with OVA (1) or OVAconjugated to various glycopolymers (2-5). B. Fluorescent images ofspleens taken from animals treated with OVA (1) or OVA conjugated tovarious glycopolymers (2-5). Formulations are as follows: 1. OVA, 2.OVA-p(Galβ-HPMA), 3. OVA-p(Gal-HPMA), 4. OVA-p(Gluβ-HPMA), 5.OVA-p(Glu-HPMA).

FIGS. 16A-16F depict data related to experiments comparing linkermoieties. OVA-p(Gal-HPMA), OVA-p(Glu-HPMA), OVA-p(Galβ-HPMA), andOVA-p(Gluβ-HPMA) conjugates were synthesized and tested for theirability to induce antigen-specific T cell anergy and eliminate the Tcell population responsible for long term memory. FIG. 16A shows aschematic of the treatment regimen for 7-day experiment. FIG. 16Bdepicts the percentage of proliferating OTI splenic T cells as assayedby CSFE dilution. FIG. 16C depicts the percentage of Annexin V+OTI Tcells in the spleens of animals treated with OVA-glycopolymer conjugatesor free OVA. FIG. 16D depicts the percentage of PD-1+ splenic OTI cells.FIG. 16E depicts the percentage of T memory cells in the OTI population,where T memory cells was defined as CD62L+ and CD44+. FIG. 16F depictsthe percentage of OTI cells expressing CD127. Of particular note is theunexpectedly enhanced efficacy of compositions employing glucose orgalactose in the 8-conformation, as compared to the α-conformation.*=P<0.05**=P<0.01; #=P<0.05, ##=P<0.01 and ###=P<0.001 (#'s representsignificance as compared to animals treated with OVA alone).

FIG. 17 depicts data related to development of symptoms of diabetes innave, control, and experimental groups.

FIG. 18 depicts the various treatment groups and the experimentaltimeline used in an experiment related to evaluation of induction oflong-lasting tolerance in transferred T cells.

FIGS. 19A-19B depict data related to T cell composition in the lymphnode. FIG. 19A depicts the remaining OTI cells after antigen challenge(as a percentage of total CD8⁺ cells) in the various treatment groups.FIG. 19B depicts the remaining OTII cells after antigen challenge (as apercentage of total CD4⁺ cells) in the various treatment groups.

FIG. 20 depicts the various treatment groups and the experimentaltimeline used in an experiment related to evaluation of induction oflong-lasting tolerance in endogenous T cells.

FIGS. 21A-21B depict data related to T cell composition in the lymphnode. FIG. 21A depicts the remaining OTI cells after antigen challenge(as a percentage of total CD8⁺ cells) in the various treatment groups.FIG. 21B depicts the remaining OTII cells after antigen challenge (as apercentage of total CD4⁺ cells) in the various treatment groups.

FIG. 22 depicts the experimental design used to evaluate the ability ofcompositions as disclosed herein to prophylactically reduce antibodyresponse.

FIG. 23 depicts experimental data related to the amount ofanti-asparaginase antibodies from mice in the various treatment groups.

FIGS. 24A-24B depict the experimental design and composition used inevaluating tolerance to myelin oligodendrocyte glycoprotein (MOG). FIG.24A shows the experimental protocol used in immunizing donor mice andtreating recipient mice. FIG. 24B shows one example of a tolerogeniccomposition in accordance with several embodiments disclosed herein.

FIGS. 25A-25B depict experimental data related to induction of toleranceagainst MOG using a first concentration of the tolerogenic composition.FIG. 25A depicts data related to delay of disease onset in the varioustreatment groups. FIG. 25B depicts data related to reduction of weightloss in the various treatment groups.

FIGS. 26A-26B depict experimental data related to induction of toleranceagainst MOG using an additional concentration of the tolerogeniccomposition. FIG. 26A depicts data related to delay of disease onset inthe various treatment groups. FIG. 26B depicts data related to reductionof weight loss in the various treatment groups.

FIGS. 27A-27E depict experimental data related to the biodistribution ofpGal and pGlu compositions in the beta conformation. FIG. 27A depictstargeting of OVA via various conjugates to LSECs in the liver. FIG. 27Bdepicts targeting of OVA via various conjugates to Kupffer cells in theliver. FIG. 27C depicts targeting of OVA via various conjugates toCD11c+ cells in the liver. FIG. 27D depicts targeting of OVA via variousconjugates to hepatocytes in the liver. FIG. 27E depicts targeting ofOVA via various conjugates to stellate cells in the liver.

DETAILED DESCRIPTION

Two known asialoglycoprotein receptors (“ASGPRs”) are expressed onhepatocytes and liver sinusoidal endothelial cells (or “LSECs”). Othergalactose/galactosamine/N-acetylgalactosamine receptors can be found invarious forms on multiple cell types [e.g., dendritic cells,hepatocytes, LSECs, and Kupffer cells]. While the molecular and cellulartargets of glucose, glucosamine and N-acetylglucosamine can be distinctfrom those of the corresponding galactose isomers, it has been foundthat the corresponding compounds of Formula 1 where Z is a glucosylatingmoiety are comparably effective in some instances, while in someembodiments disclosed herein, they are unexpectedly effective. Dendriticcells are considered “professional antigen presenting cells,” becausetheir primary function is to present antigens to the immune system forgenerating immune responses. Some cells within the liver are known to beable to present antigens, but the liver is more known to be involved intolerogenesis. The liver is understood to be a tolerogenic organ. Forexample, lower incidences of rejection are reported in cases of multipleorgan transplants when the liver is one of the organs transplanted.LSECs are much newer to the literature; consequently their role intolerogenesis and/or moderation of inflammatory immune responses is notyet widely acknowledged or well understood. However, it is becomingclear that they also can play a significant role in the induction ofantigen-specific tolerance.

One of the distinctive features of the erythrocyte surface is itsglycosylation, i.e., the presence of significant numbers of glycosylatedproteins. Indeed, the glycophorins (e.g., glycophorin A) have beenemployed as targets for erythrocyte binding. Glycophorins are proteinswith many covalently attached sugar chains, the terminus of which issialic acid. As an erythrocyte ages and becomes ripe for clearance, theterminal sialic acid of its glycophorins tends to be lost, leavingN-acetylgalactosamine at the free end. N-acetylgalactosamine is a ligandselectively received by the ASGPR associated with hepatic cells, leadingto binding of N-acetylgalactosamine-containing substances by hepaticcells and their subsequent uptake and processing in the liver.

Heretofore, it has been understood by those skilled in the art thatglycosylation of a therapeutic agent in a manner that results in hepatictargeting should be avoided due to first-pass clearance by the liverresulting in poor circulation half-life of the therapeutic agent. By thesame token, some monoclonal antibodies need to be specificallyglycosylated at ASN297 for optimal binding to their Fc receptors. It hasnow surprisingly been found, and is disclosed herein, thatgalactosylation and glucosylation can be used in a manner that inducestolerogenesis.

The present disclosure provides, in several embodiments, certaintherapeutic compositions that are targeted for delivery to (and foruptake by) the liver, particularly hepatocytes, LSECs, Kupffer cellsand/or stellate cells, more particularly hepatocytes and/or LSECs, andeven more particularly to specifically bind ASGPR. Liver-targetingfacilitates two mechanisms of treatment: tolerization and clearance.Tolerization takes advantage of the liver's role in clearing apoptoticcells and processing their proteins to be recognized by the immunesystem as “self,” as well as the liver's role in sampling peripheralproteins for immune tolerance. Clearance takes advantage of the liver'srole in blood purification by rapidly removing and breaking down toxins,polypeptides and the like. Targeting of these compositions to the liveris accomplished by a galactosylating moiety (e.g., galactose,galactosamine and N-acetylgalactosamine, particularly conjugated at C1,C2 or C6, though some embodiments involved conjugation at other or anycarbon in the molecule), by a glucosylating moiety (e.g., glucose,glucosamine and N-acetylglucosamine, particularly conjugated at C1, C2or C6, though some embodiments involved conjugation at other or anycarbon in the molecule), or by de-sialylating a polypeptide for whichsuch liver-targeting is desired. The galactosylating or glucosylatingmoiety can be chemically conjugated or recombinantly fused to anantigen, whereas desialylation exposes a galactose-like moiety on anantigen polypeptide. The antigen can be endogenous (a self-antigen) orexogenous (a foreign antigen), including but not limited to: a foreigntransplant antigen against which transplant recipients develop anunwanted immune response (e.g., transplant rejection), a foreign food,animal, plant or environmental antigen to which patients develop anunwanted immune (e.g., allergic or hypersensitivity) response, atherapeutic agent to which patients develop an unwanted immune response(e.g., hypersensitivity and/or reduced therapeutic activity), aself-antigen to which patients develop an unwanted immune response(e.g., autoimmune disease), or a tolerogenic portion (e.g., a fragmentor an epitope) thereof; these compositions are useful for inducingtolerization to the antigen. Alternatively, the galactosylating or otherliver-targeting moiety can be conjugated to an antibody, antibodyfragment or ligand that specifically binds a circulating protein orpeptide or antibody, which circulating protein or peptide or antibody iscausatively involved in transplant rejection, immune response against atherapeutic agent, autoimmune disease, and/or allergy (as discussedabove); these compositions are useful for clearing the circulatingprotein, peptide or antibody. Accordingly, the compositions of thepresent disclosure can be used for treating an unwanted immune response,e.g., transplant rejection, an immune response against a therapeuticagent, an autoimmune disease, and/or an allergy, depending on theembodiment. Also provided are pharmaceutical compositions containing atherapeutically effective amount of a composition of the disclosureadmixed with at least one pharmaceutically acceptable excipient. Inanother aspect, the disclosure provides methods for the treatment of anunwanted immune response, such as transplant rejection, response againsta therapeutic agent, autoimmune disease or allergy.

Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

The singular forms “a,” “an,” and “the” include plural referents, unlessthe context clearly indicates otherwise.

The term “about” when used in connection with a numerical value is meantto encompass numerical values within a range typically having a lowerlimit that is, e.g., 5-10% smaller than the indicated numerical valueand having an upper limit that is, e.g., 5-10% larger than the indicatednumerical value. Also included are any values within the disclosedrange, including the listed endpoints.

As used herein, an “antigen” is any substance that serves as a targetfor the receptors of an adaptive immune response, such as the T cellreceptor, major histocompatibility complex class I and II, B cellreceptor or an antibody. In some embodiments, an antigen may originatefrom within the body (e.g., “self,” “auto” or “endogenous”). Inadditional embodiments, an antigen may originate from outside the body(“non-self,” “foreign” or “exogenous”), having entered, for example, byinhalation, ingestion, injection, or transplantation, transdermally,etc. In some embodiments, an exogenous antigen may be biochemicallymodified in the body. Foreign antigens include, but are not limited to,food antigens, animal antigens, plant antigens, environmental antigens,therapeutic agents, as well as antigens present in an allografttransplant.

An “antigen-binding molecule” as used herein relates to molecules, inparticular to proteins such as immunoglobulin molecules, which containantibody variable regions providing a binding (specific binding in someembodiments) to an epitope. The antibody variable region can be presentin, for example, a complete antibody, an antibody fragment, and arecombinant derivative of an antibody or antibody fragment. The term“antigen-binding fragment” of an antibody (or “binding portion”), asused herein, refers to one or more fragments of an antibody that retainthe ability to specifically bind a target sequence. Antigen-bindingfragments containing antibody variable regions include (withoutlimitation) “Fv”, “Fab”, and “F(ab′)₂” regions, “single domainantibodies (sdAb)”, “nanobodies”, “single chain Fv (scFv)” fragments,“tandem scFvs” (V_(H)A-V_(L)A-V_(H)B-V_(L)B), “diabodies”, “triabodies”or “tribodies”, “single-chain diabodies (scDb)”, and “bi-specific T-cellengagers (BiTEs)”.

As used herein, a “chemical modification” refers to a change in thenaturally occurring chemical structure of one or more amino acids of apolypeptide. Such modifications can be made to a side chain or aterminus, e.g., changing the amino-terminus or carboxyl terminus. Insome embodiments, the modifications are useful for creating chemicalgroups that can conveniently be used to link the polypeptides to othermaterials, or to attach a therapeutic agent.

The term “comprising”, which is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps. The phrase“consisting of” excludes any element, step, or ingredient not specified.The phrase “consisting essentially of” limits the scope of describedsubject matter to the specified materials or steps and those that do notmaterially affect its basic and novel characteristics. It is understoodthat wherever embodiments are described herein with the language“comprising”, otherwise analogous embodiments described in terms of“consisting of” and/or “consisting essentially of” are also provided.When used in the claims as transitional phrases, each should beinterpreted separately and in the appropriate legal and factual context(e.g., “comprising” is considered more of an open-ended phrase while“consisting of” is more exclusive and “consisting essentially of”achieves a middle ground).

“Conservative changes” can generally be made to an amino acid sequencewithout altering activity. These changes are termed “conservativesubstitutions” or mutations; that is, an amino acid belonging to agrouping of amino acids having a particular size or characteristic canbe substituted for another amino acid. Substitutes for an amino acidsequence can be selected from other members of the class to which theamino acid belongs. For example, the nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, methionine, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such substitutions are notexpected to substantially affect apparent molecular weight as determinedby polyacrylamide gel electrophoresis or isoelectric point. Conservativesubstitutions also include substituting optical isomers of the sequencesfor other optical isomers, specifically D amino acids for L amino acidsfor one or more residues of a sequence. Moreover, all of the amino acidsin a sequence can undergo a D to L isomer substitution. Exemplaryconservative substitutions include, but are not limited to, Lys for Argand vice versa to maintain a positive charge; Glu for Asp and vice versato maintain a negative charge; Ser for Thr so that a free —OH ismaintained; and Gln for Asn to maintain a free —NH₂. Yet another type ofconservative substitution constitutes the case where amino acids withdesired chemical reactivities are introduced to impart reactive sitesfor chemical conjugation reactions, if the need for chemicalderivatization arises. Such amino acids include but are not limited toCys (to insert a sulfhydryl group), Lys (to insert a primary amine), Aspand Glu (to insert a carboxylic acid group), or specialized noncanonicalamino acids containing ketone, azide, alkyne, alkene, and tetrazineside-chains. Conservative substitutions or additions of free —NH₂ or —SHbearing amino acids can be particularly advantageous for chemicalconjugation with the linkers and galactosylating moieties of Formula 1.Moreover, point mutations, deletions, and insertions of the polypeptidesequences or corresponding nucleic acid sequences can in some cases bemade without a loss of function of the polypeptide or nucleic acidfragment. Substitutions can include, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues (including anynumber of substitutions between those listed). A variant usable in thepresent invention may exhibit a total number of up to 200 (e.g., up to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200, including any number in between those listed) changes inthe amino acid sequence (e.g., exchanges, insertions, deletions,N-terminal truncations, and/or C-terminal truncations). In severalembodiments, the number of changes is greater than 200. Additionally, inseveral embodiments, the variants include polypeptide sequences orcorresponding nucleic acid sequences that exhibit a degree of functionalequivalence with a reference (e.g., unmodified or native sequence). Inseveral embodiments, the variants exhibit about 80%, about 85%, about90%, about 95%, about 97%, about 98%, about 99% functional equivalenceto an unmodified or native reference sequence (and any degree offunctional equivalence between those listed). The amino acid residuesdescribed herein employ either the single letter amino acid designatoror the three-letter abbreviation in keeping with the standardpolypeptide nomenclature, J. Biol. Chem., (1969), 243, 3552-3559. Allamino acid residue sequences are represented herein by formulae withleft and right orientation in the conventional direction ofamino-terminus to carboxy-terminus.

The terms “effective amount” or “therapeutically effective amount” referto that amount of a composition of the disclosure that is sufficient toeffect treatment, as defined below, when administered to a mammal inneed of such treatment. This amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the particular composition of thedisclosure chosen, the dosing regimen to be followed, timing ofadministration, manner of administration and the like, all of which canreadily be determined by one of ordinary skill in the art.

An “epitope”, also known as antigenic determinant, is the segment of amacromolecule, e.g. a protein, which is recognized by the adaptiveimmune system, such as by antibodies, B cells, major histocompatibilitycomplex molecules, or T cells. An epitope is that part or segment of amacromolecule capable of binding to an antibody or antigen-bindingfragment thereof. In this context, the term “binding” in particularrelates to a specific binding. In the context of several embodiments ofthe present invention, it is preferred that the term “epitope” refers tothe segment of protein or polyprotein that is recognized by the immunesystem.

The term galactose refers to a monosaccharide sugar that exists both inopen-chain form and in cyclic form, having D- and L-isomers. In thecyclic form, there are two anomers, namely alpha and beta. In the alphaform, the C1 alcohol group is in the axial position, whereas in the betaform, the C1 alcohol group is in the equatorial position. In particular,“galactose” refers to the cyclic six-membered pyranose, more inparticular the D-isomer and even more particularly the alpha-D-form(α-D-galactopyranose) the formal name for which is(2R,3R,4S,5R,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol.Glucose is an epimer of galactose; the formal name is(2R,3R,4S,5S,6R)-6-(hydroxmethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol.The structure and numbering of galactose and glucose are shown givingtwo non-limiting examples of stereochemical illustration.

The term “galactosylating moiety” refers to a particular type ofliver-targeting moiety. Galactosylating moieties include, but are notlimited to a galactose, galactosamine and/or N-acetylgalactosamineresidue. A “glucosylating moiety” refers to another particular type ofliver-targeting moiety and includes, but is not limited to glucose,glucosamine and/or N-acetylglucosamine.

The term “liver-targeting moiety”, refers to moieties having the abilityto direct, e.g., a polypeptide, to the liver. The liver comprisesdifferent cell types, including but not limited to hepatocytes,sinusoidal epithelial cells, Kupffer cells, stellate cells, and/ordendritic cells. Typically, a liver-targeting moiety directs apolypeptide to one or more of these cells. On the surface of therespective liver cells, receptors are present which recognize andspecifically bind the liver-targeting moiety. Liver-targeting can beachieved by chemical conjugation of an antigen or ligand to agalactosylating or glucosylating moiety, desialylation of an antigen orligand to expose underlying galactosyl or glucosyl moieties, or specificbinding of an endogenous antibody to an antigen or ligand, where theantigen or ligand is: desialylated to expose underlying galactosyl orglucosyl moieties, conjugated to a galactosylating or a glucosylatingmoiety. Naturally occurring desialylated proteins are not encompassedwithin the scope of certain embodiments of the present disclosure.

The “numerical values” and “ranges” provided for the varioussubstituents are intended to encompass all integers within the recitedrange. For example, when defining n as an integer representing a mixtureincluding from about 1 to 100, particularly about 8 to 90 and moreparticularly about 40 to 80 ethylene glycol groups, where the mixturetypically encompasses the integer specified as n±about 10% (or forsmaller integers from 1 to about 25, ±3), it should be understood that ncan be an integer from about 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99,100, 105 or 110, or any between those listed, including the endpoints ofthe range) and that the disclosed mixture encompasses ranges such as1-4, 2-4, 2-6, 3-8, 7-13, 6-14, 18-23, 26-30, 42-50, 46-57, 60-78,85-90, 90-110 and 107-113 ethylene glycol groups. The combined terms“about” and “±10%” or “±3” should be understood to disclose and providespecific support for equivalent ranges wherever used.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not.

A peptide that specifically binds a particular target is referred to asa “ligand” for that target.

A “polypeptide” is a term that refers to a chain of amino acid residues,regardless of post-translational modification (e.g., phosphorylation orglycosylation) and/or complexation with additional polypeptides, and/orsynthesis into multisubunit complexes with nucleic acids and/orcarbohydrates, or other molecules. Proteoglycans therefore also arereferred to herein as polypeptides. A long polypeptide (having overabout 50 amino acids) is referred to as a “protein.” A short polypeptide(having fewer than about 50 amino acids) is referred to as a “peptide.”Depending upon size, amino acid composition and three dimensionalstructure, certain polypeptides can be referred to as an“antigen-binding molecule,” “antibody,” an “antibody fragment” or a“ligand.” Polypeptides can be produced by a number of methods, many ofwhich are well known in the art. For example, polypeptides can beobtained by extraction (e.g., from isolated cells), by expression of arecombinant nucleic acid encoding the polypeptide, or by chemicalsynthesis. Polypeptides can be produced by, for example, recombinanttechnology, and expression vectors encoding the polypeptide introducedinto host cells (e.g., by transformation or transfection) for expressionof the encoded polypeptide

As used herein, “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The term “purified” as used herein with reference to a polypeptiderefers to a polypeptide that has been chemically synthesized and is thussubstantially uncontaminated by other polypeptides, or has beenseparated or isolated from most other cellular components by which it isnaturally accompanied (e.g., other cellular proteins, polynucleotides,or cellular components). An example of a purified polypeptide is onethat is at least 70%, by dry weight, free from the proteins andnaturally occurring organic molecules with which it naturallyassociates. A preparation of a purified polypeptide therefore can be,for example, at least 80%, at least 90%, or at least 99%, by dry weight,the polypeptide. Polypeptides also can be engineered to contain a tagsequence (e.g., a polyhistidine tag, a myc tag, a FLAG® tag, or otheraffinity tag) that facilitates purification or marking (e.g., captureonto an affinity matrix, visualization under a microscope). Thus, apurified composition that comprises a polypeptide refers to a purifiedpolypeptide unless otherwise indicated. The term “isolated” indicatesthat the polypeptides or nucleic acids of the disclosure are not intheir natural environment. Isolated products of the disclosure can thusbe contained in a culture supernatant, partially enriched, produced fromheterologous sources, cloned in a vector or formulated with a vehicle,etc.

The term “random copolymer” refers to the product of simultaneouspolymerization of two or more monomers in admixture, where theprobability of finding a given monomeric unit at any given site in apolymer chain is independent of the nature of the neighboring units atthat position (Bernoullian distribution). Thus, when the variable groupidentified as W_(p) represents a random copolymer, the chain cancomprise any sequence from 2 up to about 150 W¹ and W² groups, such as:-W¹-W²-W¹-W²-; -W²-W¹-W²-W¹-; -W¹-W¹-W¹-W²-; -W¹-W¹-W²-W²-;-W¹-W²-W²-W¹-; -W¹-W²-W¹-W²-W²-W¹-W²-W¹-; -W¹-W¹-W²-W²-W¹-W²-W²-W¹-; andW²-W²-W¹-W²-W¹-W¹-W²-W²-W¹-W²-W²-W¹; ad infinitum, where Z attached tothe various W¹ groups and the W¹ and W² groups themselves can be thesame or different.

The term “sequence identity” is used with regard to polypeptide (ornucleic acid) sequence comparisons. This expression in particular refersto a percentage of sequence identity, for example at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% to the respectivereference polypeptide or to the respective reference polynucleotide.Particularly, the polypeptide in question and the reference polypeptideexhibit the indicated sequence identity over a continuous stretch of 20,30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids or over theentire length of the reference polypeptide.

“Specific binding,” as that term is commonly used in the biologicalarts, refers to a molecule that binds to a target with a relatively highaffinity as compared to non-target tissues, and generally involves aplurality of non-covalent interactions, such as electrostaticinteractions, van der Waals interactions, hydrogen bonding, and thelike. Specific binding interactions characterize antibody-antigenbinding, enzyme-substrate binding, and certain protein-receptorinteractions; while such molecules might bind tissues besides theirspecific targets from time to time, to the extent that such non-targetbinding is inconsequential, the high-affinity binding pair can stillfall within the definition of specific binding.

The term “treatment” or “treating” means any treatment of a disease ordisorder in a mammal, including:

-   -   preventing or protecting against the disease or disorder, that        is, causing the clinical symptoms not to develop;    -   inhibiting the disease or disorder, that is, arresting or        suppressing the development of clinical symptoms; and/or    -   relieving the disease or disorder, that is, causing the        regression of clinical symptoms.

The term “unwanted immune response” refers to a reaction by the immunesystem of a subject, which in the given situation is not desirable. Thereaction of the immune system is unwanted if such reaction does not leadto the prevention, reduction, or healing of a disease or disorder butinstead causes, enhances or worsens, or is otherwise associated withinduction or worsening of a disorder or disease. Typically, a reactionof the immune system causes, enhances or worsens a disease if it isdirected against an inappropriate target. Exemplified, an unwantedimmune response includes but is not limited to transplant rejection,immune response against a therapeutic agent, autoimmune disease, andallergy or hypersensitivity.

The term “variant” is to be understood as a protein (or nucleic acid)which differs in comparison to the protein from which it is derived byone or more changes in its length, sequence, or structure. Thepolypeptide from which a protein variant is derived is also known as theparent polypeptide or polynucleotide. The term “variant” comprises“fragments” or “derivatives” of the parent molecule. Typically,“fragments” are smaller in length or size than the parent molecule,whilst “derivatives” exhibit one or more differences in their sequenceor structure in comparison to the parent molecule. Also encompassed aremodified molecules such as but not limited to post-translationallymodified proteins (e.g. glycosylated, phosphorylated, ubiquitinated,palmitoylated, or proteolytically cleaved proteins) and modified nucleicacids such as methylated DNA. Also mixtures of different molecules suchas but not limited to RNA-DNA hybrids, are encompassed by the term“variant”. Naturally occurring and artificially constructed variants areto be understood to be encompassed by the term “variant” as used herein.Further, the variants usable in the present invention may also bederived from homologs, orthologs, or paralogs of the parent molecule orfrom artificially constructed variant, provided that the variantexhibits at least one biological activity of the parent molecule, e.g.,is functionally active. A variant can be characterized by a certaindegree of sequence identity to the parent polypeptide from which it isderived. More precisely, a protein variant in the context of the presentdisclosure may exhibit at least 80% sequence identity to its parentpolypeptide. Preferably, the sequence identity of protein variants isover a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 ormore amino acids. As discussed above, in several embodiments variantsexhibit about 80%, about 85%, about 90%, about 95%, about 97%, about98%, about 99% functional equivalence to an unmodified or nativereference sequence (and any degree of functional equivalence betweenthose listed).

Compositions

One aspect of the present disclosure relates to compositions,pharmaceutical formulations, and methods of treatment employing suchcompositions, as represented by Formula 1:X

Y—Z]_(m)   Formula 1where:

-   -   m is an integer from about 1 to 100, particularly from about 1        to 20, and most particularly 1 to about 10;    -   X is an antigen moiety, particularly a foreign antigen or        self-antigen against which a patient develops an unwanted immune        response, or a tolerogenic portion (e.g., a fragment or an        epitope) of such an antigen moiety;    -   Y is a linker moiety or a direct bond, or an antibody, antibody        fragment, peptide or other ligand that specifically binds X; and    -   Z is a liver-targeting moiety, in particular galactosylating or        a glucosylating moiety.        The value form in Formula 1 will depend upon the nature of X, in        that each antigen, antibody, antibody fragment or ligand will        have an individual number and density of sites (predominantly        the N-terminal amine, lysine residues and cysteine residues) to        which a linker, a galactosylating moiety or a glucosylating        moiety can be bound. Antigens having a limited number of such        sites can be derivatized, for example, at the N or C terminus,        by adding lysine or cysteine residues (optionally via a        cleavable linker, particularly a linker having an        immunoproteosome cleavage site). Generally, it is preferred to        provide an adequate degree of galactosylation/glucosylation in        compositions of Formula 1 so as to facilitate uptake by liver        cells. Pharmaceutical formulations and methods of the disclosure        can employ a cocktail of compositions of Formula 1, respectively        bearing different X moieties (e.g., several epitopes associated        with a particular unwanted immune response).

The compositions of Formula 1 include the sub-genuses where X is aforeign transplant antigen against which transplant recipients developan unwanted immune response (e.g., transplant rejection), a foreignfood, animal, plant or environmental antigen against which patientsdevelop an unwanted immune (e.g., allergic or hypersensitivity)response, a foreign therapeutic agent against which patients develop anunwanted immune response (e.g., hypersensitivity and/or reducedtherapeutic activity), or a self-antigen against which patients developan unwanted immune response (e.g., autoimmune disease); where Y is alinker of Formulae Ya through Yp; and/or where Z is galactose,galactosamine, N-acetylgalactosamine, glucose, glucosamine orN-acetylglucosamine as illustrated by Formulae 1a through 1p asdescribed below with reference to the Reaction Schemes.

In additional embodiments, in the compositions of Formula 1, X can be anantibody, antibody fragment or ligand that specifically binds acirculating protein or peptide or antibody, which circulating protein orpeptide or antibody is causatively involved in transplant rejection,immune response against a therapeutic agent, autoimmune disease,hypersensitivity and/or allergy.

Antigens

The antigen employed as X in the compositions of Formula 1 can be aprotein or a peptide, e.g. the antigen may be a complete or partialtherapeutic agent, a full-length transplant protein or peptide thereof,a full-length autoantigen or peptide thereof, a full-length allergen orpeptide thereof, and/or a nucleic acid, or a mimetic of anaforementioned antigen. A listing of any particular antigen in acategory or association with any particular disease or reaction does notpreclude that antigen from being considered part of another category orassociated with another disease or reaction.

Antigens employed in the practice of the present disclosure can be oneor more of the following:

-   -   Therapeutic agents that are proteins, peptides, antibodies and        antibody-like molecules, including antibody fragments and fusion        proteins with antibodies and antibody fragments. These include        human, non-human (such as mouse) and non-natural (i.e.,        engineered) proteins, antibodies, chimeric antibodies, humanized        antibodies, and non-antibody binding scaffolds, such as        fibronectins, DARPins, knottins, and the like.    -   Human allograft transplantation antigens against which        transplant recipients develop an unwanted immune response.    -   Self-antigens that cause an unwanted, autoimmune response. Those        skilled in the art will appreciate that while self-antigens are        of an endogenous origin in an autoimmune disease patient, the        polypeptides employed in the disclosed compositions are        typically synthesized exogenously (as opposed to being purified        and concentrated from a source of origin).    -   Foreign antigens, such as food, animal, plant and environmental        antigens, against which a patient experiences an unwanted immune        response. Those skilled in the art will appreciate that while a        therapeutic protein can also be considered a foreign antigen due        to its exogenous origin, for purposes of clarity in the        description of the present disclosure such therapeutics are        described as a separate group. Similarly, a plant or an animal        antigen can be eaten and considered a food antigen, and an        environmental antigen may originate from a plant. They are,        however, all foreign antigens. In the interest of simplicity no        attempt will be made to describe distinguish and define all of        such potentially overlapping groups, as those skilled in the art        can appreciate the antigens that can be employed in the        compositions of the disclosure, particularly in light of the        detailed description and examples.        The antigen can be a complete protein, a portion of a complete        protein, a peptide, or the like, and can be derivatized (as        discussed above) for attachment to a linker and/or        galactosylating moiety, can be a variant and/or can contain        conservative substitutions, particularly maintaining sequence        identity, and/or can be desialylated.

In the embodiments where the antigen is a therapeutic protein, peptide,antibody or antibody-like molecule, specific antigens can be selectedfrom: Abatacept, Abciximab, Adalimumab, Adenosine deaminase,Ado-trastuzumab emtansine, Agalsidase alfa, Agalsidase beta, Aldeslukin,Alglucerase, Alglucosidase alfa, α-1-proteinase inhibitor, Anakinra,Anistreplase (anisoylated plasminogen streptokinase activator complex),Antithrombin III, Antithymocyte globulin, Ateplase, Bevacizumab,Bivalirudin, Botulinum toxin type A, Botulinum toxin type B, C1-esteraseinhibitor, Canakinumab, Carboxypeptidase G2 (Glucarpidase and Voraxaze),Certolizumab pegol, Cetuximab, Collagenase, Crotalidae immune Fab,Darbepoetin-α, Denosumab, Digoxin immune Fab, Dornase alfa, Eculizumab,Etanercept, Factor Vila, Factor VIII, Factor IX, Factor XI, Factor XIII,Fibrinogen, Filgrastim, Galsulfase, Golimumab, Histrelin acetate,Hyaluronidase, Idursulphase, Imiglucerase, Infliximab, Insulin[including recombinant human insulin (“rHu insulin”) and bovineinsulin], Interferon-α2a, Interferon-α2b, Interferon-β1a,Interferon-β1b, Interferon-γ1b, Ipilimumab, L-arginase, L-asparaginase,L-methionase, Lactase, Laronidase, Lepirudin/hirudin, Mecasermin,Mecasermin rinfabate, Methoxy Natalizumab, Octreotide, Ofatumumab,Oprelvekin, Pancreatic amylase, Pancreatic lipase, Papain,Peg-asparaginase, Peg-doxorubicin HCl, PEG-epoetin-β, Pegfilgrastim,Peg-Interferon-α2a, Peg-Interferon-α2b, Pegloticase, Pegvisomant,Phenylalanine ammonia-lyase (PAL), Protein C, Rasburicase (uricase),Sacrosidase, Salmon calcitonin, Sargramostim, Streptokinase,Tenecteplase, Teriparatide, Tocilizumab (atlizumab), Trastuzumab, Type 1alpha-interferon, Ustekinumab, vW factor. The therapeutic protein can beobtained from natural sources (e.g., concentrated and purified) orsynthesized, e.g., recombinantly, and includes antibody therapeuticsthat are typically IgG monoclonal or fragments or fusions.

Particular therapeutic protein, peptide, antibody or antibody-likemolecules include Abciximab, Adalimumab, Agalsidase alfa, Agalsidasebeta, Aldeslukin, Alglucosidase alfa, Factor VIII, Factor IX,Infliximab, Insulin (including rHu Insulin), L-asparaginase, Laronidase,Natalizumab, Octreotide, Phenylalanine ammonia-lyase (PAL), orRasburicase (uricase) and generally IgG monoclonal antibodies in theirvarying formats.

Another particular group includes the hemostatic agents (Factor VIII andIX), Insulin (including rHu Insulin), and the non-human therapeuticsuricase, PAL and asparaginase.

Unwanted immune response in hematology and transplant includesautoimmune aplastic anemia, transplant rejection (generally), and Graftvs. Host Disease (bone marrow transplant rejection). In the embodimentswhere the antigen is a human allograft transplantation antigen, specificsequences can be selected from: subunits of the various MHC class I andMHC class II haplotype proteins (for example, donor/recipientdifferences identified in tissue cross-matching), and single-amino-acidpolymorphisms on minor blood group antigens including RhCE, Kell, Kidd,Duffy and Ss. Such compositions can be prepared individually for a givendonor/recipient pair.

In the embodiments where the antigen is a self-antigen, specificantigens (and the autoimmune disease with which they are associated) canbe selected from:

-   -   In type 1 diabetes mellitus, several main antigens have been        identified: insulin, proinsulin, preproinsulin, glutamic acid        decarboxylase-65 (GAD-65 or glutamate decarboxylase 2), GAD-67,        glucose-6 phosphatase 2 (IGRP or islet-specific glucose 6        phosphatase catalytic subunit related protein),        insulinoma-associated protein 2 (IA-2), and        insulinoma-associated protein 213 (IA-2(3); other antigens        include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38,        GLIMA 38, chromogranin-A, HSP-60, carboxypeptidase E,        peripherin, glucose transporter 2,        hepatocarcinoma-intestine-pancreas/pancreatic associated        protein, S100β, glial fibrillary acidic protein, regenerating        gene II, pancreatic duodenal homeobox 1, dystrophia myotonica        kinase, islet-specific glucose-6-phosphatase catalytic        subunit-related protein, and SST G-protein coupled receptors        1-5. It should be noted that insulin is an example of an antigen        that can be characterized both as a self-antigen and a        therapeutic protein antigen. For example, rHu Insulin and bovine        insulin are therapeutic protein antigens (that are the subject        of unwanted immune attack), whereas endogenous human insulin is        a self-antigen (that is the subject of an unwanted immune        attack). Because endogenous human insulin is not available to be        employed in a pharmaceutical composition, a recombinant form is        employed in certain embodiments of the compositions of the        disclosure.        -   Human insulin, including an exogenously obtained form useful            in the compositions of the disclosure, has the following            sequence (UNIPROT P01308):

(SEQ ID NO: 1) MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALYLVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPLALEGSLQKRG IVEQCCTSIC SLYQLENYCN. 

-   -   -   GAD-65, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT Q05329):

(SEQ ID NO: 2) MASPGSGFWS FGSEDGSGDS ENPGTARAWC QVAQKFTGGIGNKLCALLYG DAEKPAESGG SQPPRAAARK AACACDQKPCSCSKVDVNYA FLHATDLLPA CDGERPTLAF LQDVMNILLQYVVKSFDRST KVIDFHYPNE LLQEYNWELA DQPQNLEEILMHCQTTLKYA IKTGHPRYFN QLSTGLDMVG LAADWLTSTANTNMFTYEIA PVFVLLEYVT LKKMREIIGW PGGSGDGIFSPGGAISNMYA MMIARFKMFP EVKEKGMAAL PRLIAFTSEHSHFSLKKGAA ALGIGTDSVI LIKCDERGKM IPSDLERRILEAKQKGFVPF LVSATAGTTV YGAFDPLLAV ADICKKYKIWMHVDAAWGGG LLMSRKHKWK LSGVERANSV TWNPHKMMGVPLQCSALLVR EEGLMQNCNQ MHASYLFQQD KHYDLSYDTGDKALQCGRHV DVFKLWLMWR AKGTTGFEAH VDKCLELAEYLYNIIKNREG YEMVFDGKPQ HTNVCFWYIP PSLRTLEDNEERMSRLSKVA PVIKARMMEY GTTMVSYQPL GDKVNFFRMVISNPAATHQD IDFLIEEIER LGQDL. 

-   -   -   IGRP, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT QN9QR9):

(SEQ ID NO: 3) MDFLHRNGVLIIQHLQKDYRAYYTFLNFMSNVGDPRNIFFIYFPLCFQFNQTVGTKMIWVAVIGDWLNLIFKWILFGHRPYWWVQETQIYPNHSSPCLEQFPTTCETGPGSPSGHAMGASCVWYVMVTAALSHTVCGMDKFSITLHRLTWSFLWSVFWLIQISVCISRVFIATHFPHQVILGVIGGMLVAEAFEHTPGIQTASLGTYLKTNLFLFLFAVGFYLLLRVLNIDLLWSVPIAKKWCANPDWIHIDTTPFAGLVRNLGVLFGLGFAINSEMFLLSCRGGNNYTLSFRLLCALTSLTILQLYHFLQIPTHEEHLFYVLSFCKSASIPLTVVAFIPYSVHMLMKQSGKKSQ.

-   -   In autoimmune diseases of the thyroid, including Hashimoto's        thyroiditis and Graves' disease, main antigens include        thyroglobulin (TG), thyroid peroxidase (TPO) and thyrotropin        receptor (TSHR); other antigens include sodium iodine symporter        (NIS) and megalin. In thyroid-associated ophthalmopathy and        dermopathy, in addition to thyroid autoantigens including TSHR,        an antigen is insulin-like growth factor 1 receptor. In        hypoparathyroidism, a main antigen is calcium sensitive        receptor.    -   In Addison's Disease, main antigens include 21-hydroxylase,        17α-hydroxylase, and P450 side chain cleavage enzyme (P450scc);        other antigens include ACTH receptor, P450c21 and P450c17.    -   In premature ovarian failure, main antigens include FSH receptor        and α-enolase.    -   In autoimmune hypophysitis, or pituitary autoimmune disease,        main antigens include pituitary gland-specific protein factor        (PGSF) 1a and 2; another antigen is type 2 iodothyronine        deiodinase.    -   In multiple sclerosis, main antigens include myelin basic        protein (“MBP”), myelin oligodendrocyte glycoprotein (“MOG”) and        myelin proteolipid protein (“PLP”).        -   MBP, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT P02686):

(SEQ ID NO: 4) MGNHAGKRELNAEKASTNSETNRGESEKKRNLGELSRTTSEDNEVFGEADANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDSAATSESLDVMASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR.

-   -   -   MOG, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT Q16653):

(SEQ ID NO: 5) MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF.

-   -   -   PLP, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT P60201):

(SEQ ID NO: 6) MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF.

-   -   -   Peptides/epitopes useful in the compositions of the            disclosure for treating multiple sclerosis include some or            all of the following sequences, individually in a            composition of Formula 1 or together in a cocktail of            compositions of Formula 1:

MBP13-32: (SEQ ID NO: 7) KYLATASTMDHARHGFLPRH; MBP83-99: (SEQ ID NO: 8)ENPWHFFKNIVTPRTP; MBP111-129: (SEQ ID NO: 9) LSRFSWGAEGQRPGFGYGG;MBP146-170: (SEQ ID NO: 10) AQGTLSKIFKLGGRDSRSGSPMARR; MOG1-20:(SEQ ID NO: 11) GQFRVIGPRHPIRALVGDEV; MOG35-55: (SEQ ID NO: 12)MEVGWYRPPFSRWHLYRNGK; and PLP139-154: (SEQ ID NO: 13) HCLGKWLGHPDKFVGI.

-   -   In rheumatoid arthritis, main antigens include collagen II,        immunoglobulin binding protein, the fragment crystallizable        region of immunoglobulin G, double-stranded DNA, and the natural        and cirtullinated forms of proteins implicated in rheumatoid        arthritis pathology, including fibrin/fibrinogen, vimentin,        collagen I and II, and alpha-enolase.    -   In autoimmune gastritis, a main antigen is H+,K+-ATPase.    -   In pernicious angemis, a main antigen is intrinsic factor.    -   In celiac disease, main antigens are tissue transglutaminase and        the natural and deamidated forms of gluten or gluten-like        proteins, such as alpha-, gamma-, and omega-gliadin, glutenin,        hordein, secalin, and avenin. Those skilled in the art will        appreciate, for example, that while the main antigen of celiac        disease is alpha gliadin, alpha gliadin turns more immunogenic        in the body through deamidation by tissue glutaminase converting        alpha gliadin's glutamines to glutamic acid. Thus, while alpha        gliadin is originally a foreign food antigen, once it has been        modified in the body to become more immunogenic it can be        characterized as a self-antigen.    -   In vitiligo, a main antigen is tyrosinase, and tyrosinase        related protein 1 and 2.        -   MART1, Melanoma antigen recognized by T cells 1, Melan-A,            including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT Q16655):

(SEQ ID NO: 14) MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPP AYEKLSAEQSPPPYSP.

-   -   -   Tyrosinase, including an exogenously obtained form useful in            the compositions of the disclosure, has the following            sequence (UNIPROT P14679):

(SEQ ID NO: 15) MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRSPCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQMKNGSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDIDFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPTNPNLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTPEGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDKAANFSFRNTLEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAFVDSIFEQWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSYLEQASRIWSWLLGAAMVGAVLTALLAGLVSLLCRHKRKQLPEE KQPLLMEKEDYHSLYQSHL.

-   -   -   Melanocyte protein PMEL, gp100, including an exogenously            obtained form useful in the compositions of the disclosure,            has the following sequence (UNIPROT P40967):

(SEQ ID NO: 16) MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV.

-   -   In myasthenia gravis, a main antigen is acetylcholine receptor.    -   In pemphigus vulgaris and variants, main antigens are desmoglein        3, 1 and 4; other antigens include pemphaxin, desmocollins,        plakoglobin, perplakin, desmoplakins, and acetylcholine        receptor.    -   In bullous pemphigoid, main antigens include BP180 and BP230;        other antigens include plectin and laminin 5.    -   In dermatitis herpetiformis Duhring, main antigens include        endomysium and tissue transglutaminase.    -   In epidermolysis bullosa acquisita, a main antigen is collagen        VII.    -   In systemic sclerosis, main antigens include matrix        metalloproteinase 1 and 3, the collagen-specific molecular        chaperone heat-shock protein 47, fibrillin-1, and PDGF receptor;        other antigens include Scl-70, U1 RNP, Th/To, Ku, Jo1, NAG-2,        centromere proteins, topoisomerase I, nucleolar proteins, RNA        polymerase I, II and III, PM-Slc, fibrillarin, and B23.    -   In mixed connective tissue disease, a main antigen is U1snRNP.    -   In Sjogren's syndrome, the main antigens are nuclear antigens        SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose)        polymerase and topoisomerase, muscarinic receptors, and the        Fc-gamma receptor IIIb.    -   In systemic lupus erythematosus, main antigens include nuclear        proteins including the “Smith antigen,” SS-A, high mobility        group box 1 (HMGB1), nucleosomes, histone proteins and        double-stranded DNA (against which auto-antibodies are made in        the disease process).    -   In Goodpasture's syndrome, main antigens include glomerular        basement membrane proteins including collagen IV.    -   In rheumatic heart disease, a main antigen is cardiac myosin.    -   In autoimmune polyendocrine syndrome type 1 antigens include        aromatic L-amino acid decarboxylase, histidine decarboxylase,        cysteine sulfinic acid decarboxylase, tryptophan hydroxylase,        tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450        cytochromes P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing        receptor protein, and the type 1 interferons interferon alpha,        beta and omega.    -   In neuromyelitis optica, a main antigen is AQP4.        -   Aquaporin-4, including an exogenously obtained form useful            in the compositions of the disclosure, has the following            sequence (UNIPROT P55087):

(SEQ ID NO: 17) MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDR GEEKKGKDQSGEVLSSV.

-   -   In uveitis, main antigens include Retinal S-antigen or        “5-arrestin” and interphotoreceptor retinoid binding protein        (IRBP) or retinol-binding protein 3.        -   S-arrestin, including an exogenously obtained form useful in            the compositions of the disclosure, has the following            sequence (UNIPROT P10523):

(SEQ ID NO: 18) MAASGKTSKS EPNHVIFKKI SRDKSVTIYL GNRDYIDHVSQVQPVDGVVL VDPDLVKGKK VYVTLTCAFR YGQEDIDVIGLTFRRDLYFS RVQVYPPVGA ASTPTKLQES LLKKLGSNTYPFLLTFPDYL PCSVMLQPAP QDSGKSCGVD FEVKAFATDSTDAEEDKIPK KSSVRLLIRK VQHAPLEMGP QPRAEAAWQFFMSDKPLHLA VSLNKEIYFH GEPIPVTVTV TNNTEKTVKKIKAFVEQVAN VVLYSSDYYV KPVAMEEAQE KVPPNSTLTKTLTLLPLLAN NRERRGIALD GKIKHEDTNL ASSTIIKEGIDRTVLGILVS YQIKVKLTVS GFLGELTSSE VATEVPFRLMHPQPEDPAKE SYQDANLVFE EFARHNLKDA GEAEEGKRDK NDVDE.

-   -   -   IRBP, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT P10745):

(SEQ ID NO: 19) MMREWVLLMSVLLCGLAGPTHLFQPSLVLDMAKVLLDNYCFPENLLGMQEAIQQAIKSHEILSISDPQTLASVLTAGVQSSLNDPRLVISYEPSTPEPPPQVPALTSLSEEELLAWLQRGLRHEVLEGNVGYLRVDSVPGQEVLSMMGEFLVAHVWGNLMGTSALVLDLRHCTGGQVSGIPYIISYLHPGNTILHVDTIYNRPSNTTTEIWTLPQVLGERYGADKDVVVLTSSQTRGVAEDIAHILKQMRRAIVVGERTGGGALDLRKLRIGESDFFFTVPVSRSLGPLGGGSQTWEGSGVLPCVGTPAEQALEKALAILTLRSALPGVVHCLQEVLKDYYTLVDRVPTLLQHLASMDFSTVVSEEDLVTKLNAGLQAASEDPRLLVRAIGPTETPSWPAPDAAAEDSPGVAPELPEDEAIRQALVDSVFQVSVLPGNVGYLRFDSFADASVLGVLAPYVLRQVWEPLQDTEHLIMDLRHNPGGPSSAVPLLLSYFQGPEAGPVHLFTTYDRRTNITQEHFSHMELPGPRYSTQRGVYLLTSHRTATAAEEFAFLMQSLGWATLVGEITAGNLLHTRTVPLLDTPEGSLALTVPVLTFIDNHGEAWLGGGVVPDAIVLAEEALDKAQEVLEFHQSLGALVEGTGHLLEAHYARPEVVGQTSALLRAKLAQGAYRTAVDLESLASQLTADLQEVSGDHRLLVFHSPGELVVEEAPPPPPAVPSPEELTYLIEALFKTEVLPGQLGYLRFDAMAELETVKAVGPQLVRLVWQQLVDTAALVIDLRYNPGSYSTAIPLLCSYFFEAEPRQHLYSVFDRATSKVTEVWTLPQVAGQRYGSHKDLYILMSHTSGSAAEAFAHTMQDLQRATVIGEPTAGGALSVGIYQVGSSPLYASMPTQMAMSATTGKAWDLAGVEPDITVPMSEALSIAQDIVALRAKVPTVLQTAGKLVADNYASAELGAKMATKLSGLQSRYSRVTSEVALAEILGADLQMLSGDPHLKAAHIPENAKDRIPGIVPMQIPSPEVFEELIKFSFHTNVLEDNIGYLRFDMFGDGELLTQVSRLLVEHIWKKIMHTDAMIIDMRFNIGGPTSSIPILCSYFFDEGPPVLLDKIYSRPDDSVSELWTHAQVVGERYGSKKSMVILTSSVTAGTAEEFTYIMKRLGRALVIGEVTSGGCQPPQTYHVDDTNLYLTIPTARSVGASDGSSWEGVGVTPHVVVPAEEALARAKEMLQHNQLRVKRSPGLQDHL.

In the embodiments where the antigen is a foreign antigen against whichan unwanted immune response can be developed, such as food antigens,specific antigens can be:

-   -   from peanut: conarachin (Ara h 1), allergen II (Ara h 2),        arachis agglutinin, conglutin (Ara h 6);        -   conarachin, for example has the sequence identified as            UNIPROT Q6PSU6    -   from apple: 31 kda major allergen/disease resistance protein        homolog (Mal d 2), lipid transfer protein precursor (Mal d 3),        major allergen Mal d 1.03D (Mal d 1);    -   from milk: α-lactalbumin (ALA), lactotransferrin; from kiwi:        actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like        protein (Act d 2), kiwellin (Act d 5);    -   from egg whites: ovomucoid, ovalbumin, ovotransferrin, and        lysozyme;    -   from egg yolks: livetin, apovitillin, and vosvetin;    -   from mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2),        lipid transfer protein (Sin a 3), profilin (Sin a 4);    -   from celery: profilin (Api g 4), high molecular weight        glycoprotein (Api g 5);    -   from shrimp: Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m        2), tropomyosin fast isoform;    -   from wheat and/or other cereals: high molecular weight glutenin,        low molecular weight glutenin, alpha-, gamma- and omega-gliadin,        hordein, secalin and/or avenin;        -   peptides/epitopes useful in the compositions of the            disclosure for treating Celiac Disease include some or all            of the following sequences, individually in a composition of            Formula 1 or together in a cocktail of compositions of            Formula 1:

DQ-2 relevant, Alpha-gliadin “33-mer” native: (SEQ ID NO: 20)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFDQ-2 relevant, Alpha-gliadin “33-mer” deamidated: (SEQ ID NO: 21)LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF DQ-8 relevant, Alpha-gliadin:(SEQ ID NO: 22) QQYPSGQGSFQPSQQNPQDQ-8 relevant, Omega-gliadin (wheat, U5UA46): (SEQ ID NO: 23)QPFPQPEQPFPW

-   -   from strawberry: major strawberry allergy Fra a 1-E (Fra a 1);        and    -   from banana: profilin (Mus xp 1).

In the embodiments where the antigen is a foreign antigen against whichan unwanted immune response is developed, such as to animal, plant andenvironmental antigens, specific antigens can, for example, be: cat,mouse, dog, horse, bee, dust, tree and goldenrod, including thefollowing proteins or peptides derived from:

-   -   weeds, (including ragweed allergens amb a 1, 2, 3, 5, and 6, and        Amb t 5; pigweed Che a 2 and 5; and other weed allergens Par j        1, 2, and 3, and Par o 1);    -   grass (including major allergens Cyn d 1, 7, and 12; Dac g 1, 2,        and 5; Hol l 1.01203; Lol p 1, 2, 3, 5, and 11; Mer a 1; Pha a        1; Poa p 1 and 5);    -   pollen from ragweed and other weeds (including curly dock, lambs        quarters, pigweed, plantain, sheep sorrel, and sagebrush), grass        (including Bermuda, Johnson, Kentucky, Orchard, Sweet vernal,        and Timothy grass), and trees (including catalpa, elm, hickory,        olive, pecan, sycamore, and walnut);    -   dust (including major allergens from species Dermatophagoides        pteronyssinus, such as Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        14, 15, 18, 20, 21, and 23; from species Dermatophagoides        farina, such as Der f 1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18,        22, and 24; from species Blomia tropicalis such as Blo t 1, 2,        3, 4, 5, 6, 10, 11, 12, 13, 19, and 21; also allergens Eur m 2        from Euroglyphus maynei, Tyr p 13 from Tyrophagus putrescentiae,        and allergens Bla g 1, 2, and 4; Per a 1, 3, and 7 from        cockroach);    -   pets (including cats, dogs, rodents, and farm animals; major cat        allergens include Fel d 1 through 8, cat IgA, BLa g 2, and cat        albumin; major dog allergens include Can f 1 through 6, and dog        albumin);    -   bee stings, including major allergens Api m 1 through 12; and    -   fungus, including allergens derived from, species of Aspergillus        and Penicillium, as well as the species Alternaria alternata,        Davidiella tassiana, and Trichophyton rubrum.

As will be appreciated by those skilled in the art, a patient can betested to identify an antigen against which an unwanted immune responsehas developed, and a protein, peptide or the like can be developed basedon that antigen and incorporated as X in a composition of the presentdisclosure.

Sialated Antigens, Antibodies, Antibody Fragments

Following are non-limiting examples of antigens, antibodies, antibodyfragments having sialylation that can be removed to leave glycosylationspecifically targeting the ASGPR: follicle stimulating hormone (FSH),human chorionic gonadotropin (HCG), luteinizing hormone (LH),osteopontin, thyroid stimulating hormone (TSH), agalsidase alfa,agalsidase beta (FABRAZYME®; Genzyme), epoetin alfa and epoetin beta,follitropin alfa (GONAL-F®; Merck/Serono) and follitropin beta(FOLLISTIM®; Schering-Plough), insulin growth factor binding protein 6(IGFBP-6), lutropin alfa (LUVERIS®; Merck/Serono), transforming growthfactor β1, antithrombin (ATryn®/TROMBATE-III®; Genzyme/TalecrisBiotherapeutics), thyrotropin alfa (THYROGEN®; Genzyme), lenograstim,sargramostim (LEUKINE®; Genzyme), interleukin-3, prourokinase,lymphotoxin, C1-esterase inhibitor (Berinert®; CSL), IgG-likeantibodies, interferon beta, coagulation factor Vila (NOVOSEVEN®; NovoNordisk), coagulation factor VIII (moroctocog alfa), coagulation factorIX (nonacog alfa) (BENEFIX®; Wyeth), and the p55 tumor necrosis receptorfusion protein. (See: Byrne et al., Drug Discovery Today, Vol 12, No.7/8, pages 319-326, April 2007 and Sola et al., BioDrugs. 2010; 24(1):9-21). Pharmaceutically relevant proteins that have previously beenhyperglycosylated and can be desialylated for hepatocyte-ASGPR targetinginclude: interferon alfa and gamma, luteinizing hormone, Fv antibodyfragments, asparaginase, cholinesterase, darbepoetin alfa (AraNESP®;Amgen), thrombopoietin, leptin, FSH, IFN-α2, serum albumin, andcorifollitropin alfa.

Proteins with glycans that do not normally terminate in sialic acids,including proteins produced in bacteria or yeast (such as arginase, someinsulins, and uricase) would not be amenable to desialylation.

Those skilled in the art will appreciate that publicly availablereferences, such as UNIPROT, disclose the presence and location of sitesfor desialylation on most if not all antigens, antibodies, antibodyfragments and ligands of interest.

Antibodies and Peptide Ligands

In the embodiments employing an antibody, antibody fragment or ligand,such moieties are chosen to specifically bind a targeted circulatingprotein or peptide or antibody, and result in hepatic uptake of thecirculating targeted moiety, possibly as an adduct with the targetingmoiety, ultimately resulting in the clearance and inactivation of thecirculating targeted moiety. For example, liver-targeted Factor VIIIwill bind and clear circulating anti-Factor VIII antibodies. Proceduresfor the identification of such moieties will be familiar to thoseskilled in the art.

Linkers

The linkers employed in the compositions of the present disclosure (“Y”in Formula 1) can include N-hydroxysuccinamidyl linkers, malaemidelinkers, vinylsulfone linkers, pyridyl di-thiol-poly(ethylene glycol)linkers, pyridyl di-thiol linkers, n-nitrophenyl carbonate linkers,NHS-ester linkers, nitrophenoxy poly(ethylene glycol)ester linkers andthe like.

One particular group of linkers comprises linkers of Formula Y′-CMPbelow (where Y′ indicates the remaining portion of the linker and R⁹ andZ are as defined). More particularly, in the group of linkers includingFormula Y′-CMP, in several embodiments the R⁹ substituent is anethylacetamido group, and even more particularly the ethylacetamido isconjugated with C1 of N-acetylgalactosamine or N-acetylglucosamine.

Di-thiol-containing linkers, particularly disulfanylethylcarbamate-containing linkers (named including a free amine of X,otherwise named a “disulfanyl ethyl ester” without including the freeamine of X) are particularly advantageous in the present compositions ashaving the ability to cleave and release an antigen in its original formonce inside a cell, for example as illustrated below (where Y′ indicatesthe remaining portion of the linker and X′ and Z are as defined)

Particularly with regard to the linkers illustrated below in Formula Yathrough Formula Yp:

-   -   the left bracket “(” indicates the bond between X and Y;    -   the right or bottom bracket “)” indicates the bond between Y and        Z;    -   n is an integer representing a mixture including from about 1 to        100, particularly about 8 to 90 (e.g., 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65        70, 75, 80, 85, 90 or 95), more particularly about 40 to 80        (e.g., 39, 40, 43, 45, 46, 48, 50, 52, 53, 55, 57, 60, 62, 65,        66, 68, 70, 73, 75, 78, 80 or 81) ethylene glycol groups, where        the mixture typically encompasses the integer specified as        n±10%;    -   p is an integer representing a mixture including from about 2 to        150, particularly about 20 to 100 (e.g., 18, 19, 20, 25, 30, 35,        40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100 or 105) and        more particularly about 30 to 40 (e.g., 28, 29, 30, 31, 32, 33,        34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44), where the mixture        typically encompasses the integer specified as p±10%;    -   q is an integer representing a mixture including from about 1 to        44, particularly about 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22) and more        particularly about 4 to 12 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12        or 13), where the mixture typically encompasses the integer        specified as q±10%; and    -   R⁸ is CH₂ (“methyl”) or CH₂—CH₂—C(CH₃)(CN)        (“1-cyano-1-methyl-propyl” or “CMP”).    -   Y′ represents the remaining portion of Y (e.g., HS-PEG); and    -   W represents a polymer of the same W¹ group, or W is a copolymer        (preferably a random copolymer) of the same or different W¹ and        W² groups, where:

-   -   where:        -   p is an integer from 2 to about 150;        -   R⁹ is a direct bond, CH₂—CH₂—NHC(O) (i.e., an ethylacetamido            group or “EtAcN”) or —CH₂—CH₂—(O—CH₂—CH₂)_(t)—NH—C(O)—            (i.e., a pegylated ethylacetamido group or “Et-PEG_(t)-AcN”)        -   t is an integer from 1 to 5, (particularly 1 to 3, and more            particularly 1 or 2); and        -   R¹⁰ is an aliphatic group, an alcohol or an aliphatic            alcohol, particularly N-(2-hydroxypropyl)methylacrylamide;            and        -   Z (not shown) is galactose, glucose, galactosamine,            glucosamine, N-acetylgalactosamine or N-acetylglucosamine.

Formulae Ya through Yp

(Linkers of Formula Yn can be synthesized via certain precursors thatrender Yn particularly suitable for conjugation to hydrophobicantigens.)

The linkers shown above as Formulae Yh through Yn are synthesized asisomers that are employed without separation. For example, the linkersof Formulae Yh, Yi, Yj and Yn will be a mixture of the8,9-dihydro-1H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8yl and8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8yl structuresillustrated below:

The linkers of Formulae Yk, YL and Ym will be a mixture of the8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-8-yl and8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-9-ylstructures illustrated below:

The presence of such isomeric mixtures does not impair the functionalityof the compositions employing such linkers.

Liver-Targeting Moieties

The galactosylating moieties employed in the compositions of the presentdisclosure serve to target the compositions to liver cells (for example,specifically binding hepatocytes) and can be selected from: galactose,galactosamine or N-acetylgalactosamine. The glucosylating moietiesemployed in the compositions of the present disclosure serve to targetthe compositions to liver cells (for example, specifically bindinghepatocytes or LSECs) and can be selected from: glucose, glucosamine orN-acetylglucosamine. It has been reported that ASGPR affinity can beretained while modifying either side of galactose's C3/C4 diol anchor(Mamidyala, Sreeman K., et al., J. Am. Chem. Soc. 2012, 134, 1978-1981),therefore the points of conjugation used in several embodiments areparticularly at C1, C2 and C6.

Particular liver-targeting moieties include galactose or glucoseconjugated at C1 or C6, galactosamine or glucosamine conjugated at C2,and N-acetylgalactosamine or N-acetylglucosamine conjugated at C6. Otherparticular liver-targeting moieties include N-acetylgalactosamine orN-acetylglucosamine conjugated at C2, more particularly conjugated to alinker bearing an R⁹ substituent that is CH₂. Still other particularliver-targeting moieties include galactose, galactosamine,N-acetylgalactosamine, glucose, glucosamine or N-acetylglucosamineconjugated at C1, more particularly conjugated to a linker bearing an R⁹substituent that is an ethylacetamido group.

Nomenclature

The compositions of Formula 1 can be named using a combination of IUPACand trivial names. For example, a compound corresponding to Formula 1where X is a cyclobutyl moiety (shown instead of an antigen forillustrative purposes), Y is Formula Ya, m is 1, n is 4 and Z isN-acetylgalactosamin-2-yl or N-acetylglucosamin-2-yl:

can be named(Z)-(21-cyclobutyl-1-oxo-14(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-ylidene)triaz-1-yn-2-iumchloride, so the corresponding composition of the disclosure where X istissue transglutaminase can be named (Z)-(21-(tissuetransglutaminase)-1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-ylidene)triaz-1-yn-2-iumchloride. The corresponding composition of the disclosure where X′ istissue transglutaminase, m is 2, n is 4 and Z′ isN-acetylgalactosamin-2-yl or N-acetylglucosamin-2-yl can be named(3Z)-((tissuetransgultaminase)-1,3-diylbis(1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-yl-21-ylidene))bis(triaz-1-yn-2-ium)chloride.

In the interest of simplification, the compositions of Formula 1 can benamed using an alternative naming system by reference to X andcorrespondence to one of Formulae 1a to 1p (as illustrated in thereaction schemes) followed by recitation of the integers for variablesm, n, p and/or q, R⁸, R⁹ and identification of the galactosylatingmoiety and the position at which it is conjugated. In some embodiments,the compounds where W is a copolymer are designated by the letter of the“Y group” followed by a “prime” (e.g., F1c′) and include the number andan identification of the comonomers. Under this system, the compositionof Formula 1a where X is ovalbumin, m is 2, n is 4 and Z isN-acetylgalactosamin-2-yl can be named “F1a-OVA-m₂-n₄-2NAcGAL.” Thecorresponding composition of Formula 1a where X is ovalbumin, m is 2, nis 4 and Z is N-acetylglucosamin-2-yl can be named“F1a-OVA-m₂-n₄-2NAcGLU.”

Similarly, the following composition of Formula 1

can be named“2-((2-(((3-(3-(22-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbonothioyl)thio)-3,6,9,12-tetraoxa-20-azadocosyl)-3,9-dihydro-8H-dibenzo[V][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)carbamoyl)oxy)ethyl)disulfanyl)ethylinsulin carboxylate.” The isomer:

can be named“2-((2-(((3-(1-(22-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbonothioyl)thio)-3,6,9,12-tetraoxa-20-azadocosyl)-1,9-dihydro-8H-dibenzo[V][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)carbamoyl)-oxy)ethyl)disulfanyl)ethylinsulin carboxylate” (bold lettering highlights added for convenience inidentifying the difference between the formal names). Employing thenaming system adopted for the present disclosure, both isomers can benamed “F1n-insulin-m₁-n₁-p₁-q₄-CMP-EtAcN-1NAcGAL” (or“F1n-insulin-m₁-n₁-p₁-q₄-CMP-EtAcN-1NAcGLU” because no stereochemistryis shown for the sugar ring) where CMP indicates that R⁸ is1-cyano-1-methyl-propyl, EtAcN indicates that R⁹ is ethylacetamido and1NAcGAL indicates Z″ is N-acetylgalactosamine conjugated at C1. Absenceof the abbreviation EtAcN before the designation for Z would indicatethat R⁹ is a direct bond.

The following composition of Formula 1 exemplifies compounds where W isa copolymer:

and can be named 2-(2-(2-(2-(QQYPSGQGSFQPSQQNPQGGGSC (SEQ ID NO:26)-sulfanyl)ethoxy)ethoxy)ethoxy)ethyl6,10-bis((2-(2-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)carbamoyl)-4-cyano-13-((2-hydroxypropyl)amino)-8-((2-hydroxypropyl)carbamoyl)-4,6,8,10,12-pentamethyl-13-oxo-12-((phenylcarbonothioyl)thio)tridecanoate.Employing the naming system adopted for the present disclosure thecompound can be named “F1c′-DQ8-relevant AlphaGliadin-m₁-n₄-p₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₂-HPMA₂)”.

Preparation of the Compositions of the Disclosure

The compositions of Formula 1 can be prepared, for example, by adjustingthe procedures described in Zhu, L., et al., Bioconjugate Chem. 2010,21, 2119-2127. Syntheses of certain compositions of Formula 1 are alsodescribed below with reference to Reaction Schemes 1 to 14. Othersynthetic approaches will be apparent to those skilled in the art.

Formula 101 (below) is an alternative representation of X

where R¹ is a free surface amino (—NH₂) or thiol (—SH) moiety positionedon X's three-dimensional structure so as to be accessible forconjugation to a linker, and X′ represents the remainder of X excludingthe identified free amino group(s) [(X″ is used in the reaction schemesto represent the remainder of X excluding free thiol group(s)].Depending upon the identity of X, there will be at least one (theN-terminal amine) and can be multiple R¹ groups (predominantly fromlysine residues or cysteine residues that are not involved in disulfidebonding), as represented by m, which is an integer from about 1 to 100,more typically 1 or from about 4 to 20, and most typically 1 to about10.

Variables employed in the reaction schemes are as defined above, andadditionally include the following, which should be understood to havethese meanings absent any specific indication otherwise with respect toa particular reaction scheme or step.

-   -   R² is OH or a protecting group;    -   R³ is OH, NH₂, NHAc, a protecting group or NH-protecting group;    -   R⁴ is OH or a protecting group;    -   R⁵ is OH or a protecting group;    -   R⁶ is OH or a protecting group;    -   Z′ is galactose or glucose conjugated at C1 or C6, galactosamine        or glucosamine conjugated at C2, or N-acetylgalactosamine        conjugated or N-acetylglucosamine at C6;    -   R⁸ is —CH₂— or —CH₂—CH₂—C(CH₃)(CN)—; and    -   R⁹ is a direct bond and Z″ is N-acetylgalactosamine conjugated        at C2; or    -   R⁹ is an ethylacetamido or a pegylated ethylacetamido group and        Z″ is galactose, glucose, galactosamine, glucosamine,        N-acetylgalactosamine or N-acetylglucosamine conjugated at C1.

Synthetic Reaction Parameters

The terms “solvent”, “inert organic solvent” or “inert solvent” mean asolvent inert under the conditions of the reaction being described inconjunction therewith [including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, pyridine and the like]. Unless specified to the contrary, thesolvents used in the reactions of the present disclosure are inertorganic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, centrifugal size exclusion chromatography,high-performance liquid chromatography, recrystallization, sublimation,fast protein liquid chromatography, gel electrophoresis, dialysis, or acombination of these procedures. Specific illustrations of suitableseparation and isolation procedures can be had by reference to theexamples hereinbelow. However, other equivalent separation or isolationprocedures can, of course, also be used.

Unless otherwise specified (including in the examples), all reactionsare conducted at standard atmospheric pressure (about 1 atmosphere) andambient (or room) temperature (about 20° C.), at about pH 7.0-8.0.

Characterization of reaction products can be made by customary means,e.g., proton and carbon NMR, mass spectrometry, size exclusionchromatography, infrared spectroscopy, gel electrophoresis.

Reaction Scheme 1 illustrates the preparation of compositions of Formula1 where Z can be galactose, glucose, galactosamine, glucosamine,N-acetylgalactosamine or N-acetylglucosamine. In that regard and asdefined above, Z′ as employed in Reaction Scheme 1 encompasses galactoseor glucose conjugated at C1 and C6 and corresponding to the followingstructures according to Formula 1:

galactosamine or glucosamine conjugated at C2 and corresponding to thefollowing structure according to Formula 1:

and N-acetylgalactosamine or N-acetylglucosamine conjugated at C6 andcorresponding to the following structure according to Formula 1:

As illustrated above in Reaction Scheme 1, Step 1, surface thiolgroup(s) can be generated on an antigen, antibody, antibody fragment orligand having free surface amino group(s) (Formula 101′) by contact witha Traut reagent (Formula 102) at a pH of about 8.0 for about 1 hour togive the Formula 103′, from which unreacted Traut's reagent is removed,e.g., via centrifugal size exclusion chromatography. The two structuresshown below, illustrate the product of Reaction Scheme 1, Step 1,respectively showing the free surface amino group(s) originally found onX (i.e., Formula 103′ where X′ represents the remainder of X excludingthe identified free surface amino groups) and omitting the free surfaceamino group(s) (i.e., Formula 103). This parallels the distinctionillustrated as between X and Formula 101. The convention has beenfollowed in the subsequent reaction schemes.

In Reaction Scheme 1, Step 2, a pyridyl di-thiol-poly(ethyleneglycol)-NHS ester (Formula 104) is contacted with galactosamine orglucosamine (Formula 105 where R³ is NH₂ and R², R⁴, R⁵ and R⁶ are OH)with stirring at about pH 8 for about 1 hour to give the correspondingpyridyl di-thiol-poly(ethylene glycol)-sugar of Formula 106A, which canbe used without further purification.

In Reaction Scheme 1, Step 3, 4,4′-dithiodipyridine (Formula 107) iscontacted with a thiol-poly(ethylene glycol)propanoic acid (Formula 108)to give the corresponding pyridyl di-thiol-poly(ethyleneglycol)propanoic acid (Formula 109).

In Reaction Scheme 1, Step 4, the acid of Formula 109 is contacted witha protected galactose or N-acetylgalactosamine of Formula 105 where R²is OH and R³, R⁴, R⁵ and R⁶ are protecting groups (“PG”), where R⁶ is OHand R², R³, R⁴ and R⁵ are PG, or where R⁶ is N-acetyl and R², R³, R⁴ andR⁵ are PG to give the corresponding pyridyl di-thiol-poly(ethyleneglycol)-sugars of Formulae 106B, 106C and 106D, which can be usedfollowing de-protection.

In Reaction Scheme 1, Step 5, to a stirred solution of the product ofStep 1 (Formula 103′) is added an excess (corresponding to the value ofm) of the product of Step 2 or Step 4 (Formula 106, i.e., 106A, 106B,106C or 106D) for about 1 hour, followed by centrifugal sized exclusionchromatography to remove any free remaining reactants to yield thecorresponding product according to Formula 1a, respectively, Formula1aA, Formula 1aB, Formula 1aC and Formula 1aD.

The compositions corresponding to Formula 1a can be named, respectively,e.g., as follows:

-   -   “F1aA-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-2NGAL”    -   “F1aB-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-1GAL”    -   “F1aC-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-6GAL”    -   “F1aD-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-6NAcGAL”    -   “F1aA-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-2NGLU”    -   “F1aB-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-1GLU”    -   “F1aC-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-6GLU”    -   “F1aD-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-6NAcGLU”        respectively, for products made employing an intermediate        according to Formulae 106A-D.

Reaction Schemes 2-14 illustrate preparation of the compounds where W isa polymer of the same W¹ group. For the purposes of the nomenclatureemployed therewith, except as expressly stated otherwise, Z″ refers toN-acetylgalactosamine or N-acetylglucosamine conjugated at C2:

or to galactose, glucose, galactosamine, glucosamine,N-acetylgalactosamine or N-acetylglucosamine conjugated at C1. It shouldbe noted that, according to several embodiments, in order to improveyields, the C1 conjugated compositions can be protected duringsynthesis, for example by cyclizing the amine with the C3 hydroxyl andde-protecting following incorporation of the protected galactosamineinto the adjacent portion of the linker.

The poly(galactose methacrylate) and poly(glucose methacrylate)reactants of Formulae 201, 401, 501, 601, 701, 803 and 1401 can beprepared by methacrylating galactose or glucose, e.g., contactinggalactosamine or glucosamine and methacrylate anhydride, followed byreversible addition-fragmentation chain transfer (RAFT) polymerizationwith a corresponding RAFT agent in the presence ofazobisisobutyronitrile (AIBN) in a suitable solvent, starting withfreeze-thaw cycles followed by heating at about 60-80° C., preferably70° C. for about 5-8, preferably about 6 hours. The polymer can beprecipitated in a lower alkanol, preferably methanol.

As illustrated in Reaction Scheme 2, an antigen, antibody, antibodyfragment or ligand having free surface thiol group(s) prepared, e.g., asdescribed with reference to Reaction Scheme 1, Step 1 (Formula 103′) iscontacted with an excess (corresponding to the value of m) of a pyridyldi-thiol-poly(ethylene glycol) of Formula 201 for about 1 hour to yieldthe corresponding product according to Formula 1b.

The compositions of Formula 1b can be named as follows:

-   -   “F1b-X′-m_(m)-n_(n)-p_(p)-2NAcGAL”        “F1b-X′-m_(m)-n_(n)-p_(p)-2NAcGLU” or        “F1b-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”. For example, the composition        of Formula 1b where X′ is uricase, m is 1, n is 4, p is 4 and Z″        is N-acetylgalactosamine conjugated at C2 can be named        “F1b-uricase-m₁-n₄-p₄-2NAcGAL” or        “30-(uricase)-3,5,7,9-tetramethyl-12-oxo-1-phenyl-1-thioxo-3,5,7,9-tetrakis((2,4,5-trihydroxy-6-(hydrownethyl)tetrahydro-2H-pyran-3-yl)carbamoyl)-13,16,19,22-tetraoxa-2,25,26-trithiatriacontan-30-iminium”.

As illustrated in Reaction Scheme 3, an antigen, antibody, antibodyfragment or ligand having native free surface thiol group(s) (cysteines)[Formula 101″ corresponding to Formula 101 and illustrating where X″, asthe term will be subsequently employed, represents X excluding theidentified free surface thiol group(s)] is contacted with an excess(corresponding to the value of m) of a pyridyl di-thiol-poly(ethyleneglycol) of Formula 201 to yield the corresponding product according toFormula 1c.

The compositions corresponding to Formula 1c can be named as follows:

-   -   “F1c-X′-m_(m)-n_(n)-p_(p)-2NAcGAL”        “F1c-X′-m_(m)-n_(n)-p_(p)-2NAcGLU” or        “F1c-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 4, an antigen, antibody, antibodyfragment or ligand having native free surface thiol group(s) of Formula101″ is contacted with an excess (corresponding to the value of m) of apyridyl di-thiol of Formula 401 to yield the corresponding productaccording to Formula 1d.

The compositions corresponding to Formula 1d can be named as follows:

-   -   “F1d-X′-m_(m)-p_(p)-2NAcGAL” “F1d-X′-m_(m)-p_(p)-2NAcGLU” or        “F1d-X′-m_(m)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 5, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) of Formula101′ is contacted with an excess (corresponding to the value of m) of an-nitrophenyl carbonate of Formula 501 to yield the correspondingproduct according to Formula 1e.

The compositions corresponding to Formula 1e can be named as follows:

-   -   “F1e-X′-m_(m)-p_(p)-2NAcGAL” “F1e-X′-m_(m)-p_(p)-2NAcGLU” or        “F1e-X′-m_(m)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 6, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) of Formula101′ is contacted with an excess (corresponding to the value of m) of an-nitrophenyl carbonate poly(ethylene glycol)ester of Formula 601 toyield the corresponding product according to Formula 1f.

The compositions corresponding to Formula 1f can be named as follows:

-   -   “F1f-X′-m_(m)-n_(n)-p_(p)-2NAcGAL”        “F1f-X′-m_(m)-n_(n)-p_(p)-2NAcGLU” or        “F1f-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 7, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) of Formula101′ is contacted with an excess (corresponding to the value of m) of aNHS-ester poly(ethylene glycol)ester of Formula 701 to yield thecorresponding product according to Formula 1g.

The compositions corresponding to Formula 1g can be named as follows:

-   -   “F1g-X′-m_(m)-p_(p)-2NAcGAL” “F1g-X′-m_(m)-p_(p)-2NAcGLU” or        “F1g-X′-m_(m)-p_(p)-EtAcN-Z”

As illustrated in Reaction Scheme 8, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is contacted with an excess (corresponding to the value ofm) of an amine-reactive linker for Click chemistry of Formula 801 toyield the corresponding product according to Formula 802.

In Reaction Scheme 8, Step 2, the product of Formula 802 is thencontacted with an equivalent amount (again corresponding to the value ofm) of a galactos(amine) polymer of Formula 803 to yield thecorresponding isomeric product according to Formula 1h. The two isomers,illustrated above, result from non-specific cyclization of the azide ofFormula 803 with the triple bond of Formula 802. Such non-specificcyclization occurs in the synthesis of other compositions where Y isselected from Formulae Yh through Yn, but will not be illustrated ineach instance.

The compositions corresponding to Formula 1h can be named as follows:

-   -   “F1h-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL”        “F1h-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGLU” or        “F1h-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

As illustrated in Reaction Scheme 9, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface thiol group(s) ofFormula 101″ is contacted with an excess (corresponding to the value ofm) of a thiol-reactive linker for Click chemistry of Formula 901 toyield the corresponding product according to Formula 902″.

In Reaction Scheme 9, Step 2, the product of Formula 902″ is thencontacted with an equivalent amount (again corresponding to the value ofm) of a galactos(amine) polymer of Formula 803 to yield thecorresponding isomeric product according to Formula 1i.

The compositions corresponding to Formula 1i can be named as follows:

-   -   “F1i-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL”        “F1i-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGLU” or        “F1i-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

By following the procedures described with regard to Reaction Scheme 9,but substituting starting material 101″ with a compound of Formula 103′(derivatized with the Traut reagent) there is obtained the correspondingisomeric product of Formula 1j as shown below.

The compositions corresponding to Formula 1j can be named as follows:

-   -   “F1j-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL”        “F1j-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGLU” or        “F1j-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

As illustrated in Reaction Scheme 10, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface thiol group(s) ofFormula 101″ is contacted with an excess (corresponding to the value ofm) of a thiol-reactive linker for Click chemistry of Formula 1001 toyield the corresponding product according to Formula 1002.

In Reaction Scheme 10, Step 2, the product of Formula 1002 is thencontacted with an equivalent amount (again corresponding to the value ofm) of a galactos(amine) polymer of Formula 803 to yield thecorresponding isomeric product according to Formula 1k.

The compositions corresponding to Formula 1k can be named as follows:

-   -   “F1k-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL”        “F1k-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGLU” or        “F1k-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

By following the procedures described with regard to Reaction Scheme 10,but substituting starting material 101″ with a compound of Formula 103′(derivatized with the Traut reagent) there is obtained the correspondingisomeric product of Formula 1L as shown below.

The compositions corresponding to Formula 1L can be named as follows:

-   -   “F1L-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL”        “F1L-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGLU” or        “F1L-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

As illustrated in Reaction Scheme 11, Step 1, galactose, protectedgalactosamine or N-Acetyl-D-galactosamine (Formula 1101 where R³ and R⁴are OH, R³ is NH-protecting group (e.g., cyclized with R⁴) or R³ is NHAcand R⁴ is OH, respectively) is contacted with 2-chloroethan-1-olfollowed by cooling and the dropwise addition of acetylchloride. Thesolution is warmed to room temperature and then heated to 70° C. forseveral hours. Ethanol is added to the crude product and the resultingsolution is stirred in the presence of carbon and then filtered followedby solvent removal to yield the corresponding product of Formula 1102.

As illustrated in Reaction Scheme 11, Step 2, the product of Formula1102 is added to an excess of sodium azide and heated to 90° C. forseveral hours, then filtered followed by solvent removal to yield thecorresponding product of Formula 1103.

As illustrated in Reaction Scheme 11, Step 3, the product of Formula1103 is added to a solution of palladium on carbon and ethanol, andstirred under hydrogen gas (3 atm) for several hours, then filteredfollowed by solvent removal to yield the corresponding product ofFormula 1104.

As illustrated in Reaction Scheme 11, Step 4, the product of Formula1104 is added to a solution of methacrylate anhydride. Triethylamine isadded and the reaction stirred for 2 hours followed by solvent removaland isolation to yield the corresponding product of Formula 1105.

As illustrated in Reaction Scheme 11, Step 5, an azide-modified uRAFTagent (Formula 1106) is added to a solution of the product of Formula1105 with azobisisobutyronitrile, subjected to 4 free-pump-thaw cyclesand then stirred at 70° C. After several hours the corresponding polymerproduct of Formula 1107 is precipitated by addition of a lower alkanolfollowed by solvent removal. Where R³ is NH-protecting group (e.g.,cyclized with R⁴) the protecting group(s) is(are) removed at this point.

As illustrated in Reaction Scheme 11, Step 6, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is added to a pH 8.0 buffer and contacted with an excess(corresponding to the value of m) of a dioxopyrrolidine of Formula 1108with stirring. After 1 hour, unreacted Formula 1108 is removed and theresulting product of Formula 1109 is used without further purification.

As illustrated in Reaction Scheme 11, Step 7, the product of Formula1107 is added to a pH 8.0 buffer, to which is added the product ofFormula 1109. After stirring for 2 hours, the excess Formula 1107 isremoved to yield the corresponding isomeric product of Formula 1m.

By substitutingN-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)methacrylamidefor the product of Formula 1105 in Step 5 and continuing with Steps 6and 7, the corresponding isomeric product of Formula 1m where Z″ isN-acetylgalactosamine conjugated at C2 are obtained.

The compositions corresponding to Formula 1m can be named as follows:

-   -   “F1m-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z” where Z″ is 1GAL,        1NGAL, 1 NAcGAL, “F1m-X′-m_(m)-n_(n)-p_(p)-q_(q)-2 NAcGAL” or        “F1m-X′-m_(m)-n_(n)-p_(p)-q_(q)-2 NAcGLU” (or the corresponding        1GAL, 1 GLU, 1NGAL, 1NGLU, 1NAcGAL or 1NAcGLU compounds).

The synthetic approach of Reaction Scheme 12 is particularly suitablefor hydrophobic antigens, antibodies, antibody fragments and ligands(e.g., Insulin) due to the use of organic solvents.

As illustrated in Reaction Scheme 12, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is dissolved in an organic solvent (e.g., DMF) containingtriethylamine. To this is added an amount (corresponding to the value ofm) of a compound of Formula 1201 followed by stirring and the additionof t-butyl methyl ether. The corresponding product of Formula 1202 isrecovered as a precipitate.

The product of Formula 1202 is resuspended in the organic solvent and anamount (corresponding to the value of m) of Formula 1107 (obtained,e.g., as described with reference to Reaction Scheme 11) is addedfollowed by stirring. The reaction product is precipitated via theaddition of dichloromethane, followed by filtration and solvent removal.Purification (e.g., resuspension in PBS followed by centrifugal sizeexclusion chromatography yields the corresponding isomeric product ofFormula 1n.

The compositions corresponding to Formula 1n can be named as follows:

-   -   “F1n-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z” where Z″ is 1GAL,        1NGAL, 1NAcGAL, 1GLU, 1NGLU, 1NAcGLU, or        “F1m-X′-n_(m)-n_(n)-p_(p)-q_(q)-2 NAcGAL” or        “F1m-X′-n_(m)-n_(n)-p_(p)-q_(q)-2 NAcGLU”.

In Reaction Scheme 13, Step 1, a nitrophenoxycarbonyl-oxyalkyldi-thiol-poly(ethylene glycol)-NHS ester (Formula 1301) is contactedwith galactose, galactosamine or N-acetylgalactosamine (Formula 105) togive the corresponding product of Formula 1302, along with the other twoillustrated products, from which the desired nitrophenoxycarbonyldi-thiol-poly(ethylene glycol)-carboxyethyl galactose, galactosamine orN-acetylgalactosamine of Formula 1302 is isolated before proceeding tothe next step.

As illustrated in Reaction Scheme 13, Step 2, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is contacted with an excess (corresponding to the value ofm) of the product of Formula 1302 to yield the corresponding productaccording to Formula 1o.

The compositions corresponding to Formula 1o can be named as follows:

-   -   “F1o-X′-m_(m)-n_(n)-Z′.”

As illustrated in Reaction Scheme 14, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) (Formula101′) is contacted with an excess (corresponding to the value of m) of apyridyl di-thiol-poly(ethylene glycol)-NHS ester of Formula 1401 toyield the corresponding product according to Formula 1p.

The compositions corresponding to Formula 1p can be named as follows:

-   -   “F1p-X′-m_(m)-n_(n)-p_(p)-2NAcGAL”        “F1p-X′-m_(m)-n_(n)-p_(p)-2NAcGLU” or        “F1p-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”.

Reaction Schemes 15-18 illustrate preparation of the compounds where Wis a copolymer of the same or different W¹ and W² groups.

As illustrated in Reaction Scheme 15, Step 1, galactose or glucose(Formula 1101 where R³ and R⁴ are OH), protected galactosamine orprotected glucosamine (Formula 1101 where R³ is NH-protecting group,e.g., cyclized with R⁴) or N-acetyl-D-galactosamine orN-acetyl-D-glucosamine (Formula 1101 where R³ is NHAc and R⁴ is OH) iscontacted with a 2-(poly-(2-chloroethoxy)ethoxy)ethan-1-ol of Formula1501 (where t is 1 to 5) followed by cooling and the dropwise additionof acetylchloride. The solution is warmed to room temperature and thenheated to 70° C. for several hours. Ethanol is added to the crudeproduct and the resulting solution is stirred in the presence of carbonand then filtered followed by solvent removal to yield the correspondingproduct of Formula 1502.

As illustrated in Reaction Scheme 15, Step 2, the product of Formula1502 is added to an excess of sodium azide and heated to 90° C. forseveral hours, then filtered followed by solvent removal to yield thecorresponding product of Formula 1503.

As illustrated in Reaction Scheme 15, Step 3, the product of Formula1503 is added to a solution of palladium on carbon and ethanol, andstirred under hydrogen gas (3 atm) for several hours, then filteredfollowed by solvent removal to yield the corresponding product ofFormula 1504.

As illustrated in Reaction Scheme 15, Step 4, the product of Formula1504 is added to a solution of methacrylate anhydride. Triethylamine isadded and the reaction stirred for 2 hours followed by solvent removaland isolation to yield the corresponding product of Formula 1505.Alternatively, pentafluorophenyl methacrylate (or another acrylatingagent) can be used to prepare the corresponding product of Formula 1505.In some embodiments, the product of formula 1504 is added to DMF.Triethyl amine (e.g., an organic base) is added and the mixture iscooled (e.g., to 4° C. using an ice bath). Subsequently,pentafluorophenyl methacrylate (or another acrylating agent) is added(e.g., drop-wise with constant stirring). After a period of time (e.g.,30 minutes), the cooling (e.g., ice-bath) is removed and the reaction isallowed to stir at room temperature for a period of time (e.g., 4hours). In some embodiments, the solvent is then removed. In someembodiments, the product is purified using flash chromatography.

As illustrated in Reaction Scheme 15, Step 5, an azide-modified uRAFTagent of Formula 1106 and a methacrylamide of Formula 1506 are added toa solution of the product of Formula 1505 with azobisisobutyronitrile,subjected to 4 free-pump-thaw cycles and then stirred at 70° C. Afterseveral hours the corresponding random copolymer product of Formula 1507is precipitated by addition of a lower alkanol or acetone followed bysolvent removal. Where R³ is NH-protecting group (e.g., cyclized withR⁴) the protecting group(s) is(are) removed at this point.

As illustrated in Reaction Scheme 15, Step 6, the product of Formula1507 is added to a pH 8.0 buffer, to which is added the product ofFormula 1109 (prepared, for example, as described with reference toReaction Scheme 11). After stirring for 2 hours, the excess Formula 1109is removed to yield the corresponding isomeric random copolymer productof Formula 1m′.

By adding more than one methacrylamide of Formula 1505 in Step 5 (forexample, glucose and galactose methacrylamides, or two or moremethacrylamides having different values for t) and/or two or moremethacrylamides of Formula 1506, and continuing with Step 6, thecorresponding product of Formula 1m′ having a mixture of R³ and/or PEG(“t”) and/or R¹⁰ groups, i.e., compounds of Formula 1 where W is arandom copolymer of different W¹ and W² groups are obtained.

The compositions corresponding to Formula 1m′ can be named as follows:

-   -   “F1m′-X′-m_(m)-n_(n)-p_(p)-q_(q)-R⁸-poly-(W¹ _(t)Z″_(W1p-ran-)W²        _(W2p))”.

As illustrated in Reaction Scheme 16, Step 1, a compound of Formula 1601is contacted with compounds of Formulae 1505 and 1506 under conditionsanalogous to those of Reaction Scheme 15, Step 5, to afford thecorresponding compound of Formula 1602.

In some embodiments, the following synthesis is performed to formcompound 1601a (an embodiment of 1601):

In some embodiments, t is an integer from about 1 to about 10 or about 1to about 5. In some embodiments, an oligoethylene glycol (1650) isreacted with p-toluenesulfonyl chloride (or some other agent capable offunctionalizing 1650 with a leaving group) to form oligoethylene glycolmono p-toluenesulfonate (1651) (or some other oligoethylene glycolfunctionalized with a leaving group). In some embodiments, compound 1651can be reacted with potassium thioacetate to form compound 1652. In someembodiments, compound 1652 is reacted with 2,2-dithiodipyridine to formcompound 1653. In some embodiments, compound 1653 is coupled to compound1654 to form compound 1601a.

As illustrated in Reaction Scheme 16, Step 2, the compound of Formula1602 is contacted with a compound of Formula 101″ under conditionsanalogous to those of Reaction Scheme 15, Step 6, to afford thecorresponding compound of Formula 1c′.

The compositions corresponding to Formula 1c′ can be named as follows:

-   -   “F1c′-X′-m_(m)-n_(n)-p_(p)-R⁸-poly-(W¹ _(t)Z″-ran-W²)”.

As illustrated in Reaction Scheme 17, Step 1, a compound of Formula 600′is contacted with compounds of Formulae 1505 and 1506 under conditionsanalogous to those of Reaction Scheme 15, Step 5, to afford thecorresponding compound of Formula 601′.

As illustrated in Reaction Scheme 17, Step 2, the compound of Formula601′ is contacted with a compound of Formula 101′ under conditionsanalogous to those of Reaction Scheme 15, Step 6, to afford thecorresponding compound of Formula 1f.

The compositions corresponding to Formula 1f can be named as follows:

-   -   “F1f-X′-m_(m)-n_(n)-p_(p)-R⁸-poly-(W¹ _(t)Z″-ran-W²)”.

As illustrated in Reaction Scheme 18, Step 1, a compound of Formula 700′is contacted with compounds of Formulae 1505 and 1506 under conditionsanalogous to those of Reaction Scheme 15, Step 5, to afford thecorresponding compound of Formula 701′.

As illustrated in Reaction Scheme 18, Step 2, the compound of Formula701′ is contacted with a compound of Formula 101′ under conditionsanalogous to those of Reaction Scheme 15, Step 6, to afford thecorresponding compound of Formula 1g′.

The compositions corresponding to Formula 1g′ can be named as follows:

-   -   “F1g′-X′-m_(m)-p_(p)-R⁸-poly-(W¹ _(t)Z″-ran-W²)”.

Particular Processes and Last Steps

A compound of Formula 103′ is contacted with an excess (corresponding tothe value of m) of a compound of Formula 106 to give the correspondingproduct of Formula 1a.

A compound of Formula 103′ is contacted with an excess (corresponding tothe value of m) of a compound of Formula 201 to give the correspondingproduct of Formula 1b.

A compound of Formula 802, 902 or 1002 is contacted with an excess(corresponding to the value of m) of a compound of Formula 803 to givethe corresponding product of Formula 1h, Formula 1i or Formula 1k,respectively.

A compound of Formula 1109 is contacted with an excess (corresponding tothe value of m) of a compound of Formula 1107 to give the correspondingproduct of Formula 1m, particularly where n is about 80, p is about 30,q is about 4, and m being a function of the antigen is about 2 to 10.

A compound of Formula 1202 is contacted with an excess (corresponding tothe value of m) of a compound of Formula 1107 to give the correspondingproduct of Formula 1n, particularly where n is about 1, p is about 30, qis about 4, and m being a function of the antigen is about 2 to 10.

A compound of Formula 1507 is contacted with a compound of Formula 1109to give the corresponding product of Formula 1m′, particularly where nis about 4, p is about 90, q is about 4, t is about 1 or 2, R³ is NHAc,R⁴ is OH, R⁸ is CMP, R¹⁰ is 2-hydroxypropyl and m being a function ofthe antigen is about 1 to 10.

A compound of Formula 101″ is contacted with a compound of Formula 1602to give the corresponding product of Formula 1c′, particularly where nis about 4, p is about 90, t is about 1 or 2, R³ is NHAc, R⁴ is OH, R⁸is CMP, R¹⁰ is 2-hydroxypropyl and m being a function of the antigen isabout 1 to 10.

A compound of Formula 101′ is contacted with a compound of Formula 601′to give the corresponding product of Formula 1f′, particularly where nis about 4, p is about 90, t is about 1 or 2, R³ is NHAc, R⁴ is OH, R⁸is CMP, R¹⁰ is 2-hydroxypropyl and m being a function of the antigen isabout 1 to 10.

A compound of Formula 101′ is contacted with a compound of Formula 701′to give the corresponding product of Formula 1g′, particularly where nis about 4, p is about 90, t is about 1 or 2, R³ is NHAc, R⁴ is OH, R⁸is CMP, R¹⁰ is 2-hydroxypropyl and m being a function of the antigen isabout 1 to 10.

Particular Compositions

By way of non-limiting example, a particular group preferred for thecompositions, pharmaceutical formulations, methods of manufacture anduse of the present disclosure are the following combinations andpermutations of substituent groups of Formula 1 (sub-grouped,respectively, in increasing order of preference):

-   -   X is a foreign transplant antigen against which transplant        recipients develop an unwanted immune response, a foreign        antigen to which patients develop an unwanted immune response, a        therapeutic protein to which patients develop an unwanted immune        response, a self-antigen to which patients develop an unwanted        immune response, or a tolerogenic portion thereof.    -   X is a therapeutic protein to which patients develop an unwanted        immune response selected from: Abatacept, Abciximab, Adalimumab,        Adenosine deaminase, Ado-trastuzumab emtansine, Agalsidase alfa,        Agalsidase beta, Aldeslukin, Alglucerase, Alglucosidase alfa,        α-1-proteinase inhibitor, Anakinra, Anistreplase (anisoylated        plasminogen streptokinase activator complex), Antithrombin III,        Antithymocyte globulin, Ateplase, Bevacizumab, Bivalirudin,        Botulinum toxin type A, Botulinum toxin type B, C1-esterase        inhibitor, Canakinumab, Carboxypeptidase G2 (Glucarpidase and        Voraxaze), Certolizumab pegol, Cetuximab, Collagenase,        Crotalidae immune Fab, Darbepoetin-α, Denosumab, Digoxin immune        Fab, Dornase alfa, Eculizumab, Etanercept, Factor Vila, Factor        VIII, Factor IX, Factor XI, Factor XIII, Fibrinogen, Filgrastim,        Galsulfase, Golimumab, Histrelin acetate, Hyaluronidase,        Idursulphase, Imiglucerase, Infliximab, Insulin (including rHu        insulin and bovine insulin), Interferon-α2a, Interferon-α2b,        Interferon-β1a, Interferon-β1b, Interferon-γ1b, Ipilimumab,        L-arginase, L-asparaginase, L-methionase, Lactase, Laronidase,        Lepirudin/hirudin, Mecasermin, Mecasermin rinfabate, Methoxy        Ofatumumab, Natalizumab, Octreotide, Oprelvekin, Pancreatic        amylase, Pancreatic lipase, Papain, Peg-asparaginase,        Peg-doxorubicin HCl, PEG-epoetin-6, Pegfilgrastim,        Peg-Interferon-α2a, Peg-Interferon-α2b, Pegloticase,        Pegvisomant, Phenylalanine ammonia-lyase (PAL), Protein C,        Rasburicase (uricase), Sacrosidase, Salmon calcitonin,        Sargramostim, Streptokinase, Tenecteplase, Teriparatide,        Tocilizumab (atlizumab), Trastuzumab, Type 1 alpha-interferon,        Ustekinumab, and vW factor.        -   Especially where X is Abciximab, Adalimumab, Agalsidase            alfa, Agalsidase beta, Aldeslukin, Alglucosidase alfa,            Factor VIII, Factor IX, Infliximab, L-asparaginase,            Laronidase, Natalizumab, Octreotide, Phenylalanine            ammonia-lyase (PAL), or Rasburicase (uricase).            -   Particularly where X is Factor VIII, Factor IX, uricase,                PAL or asparaginase.    -   X is a self-antigen polypeptide selected for treating type 1        diabetes mellitus, pediatric multiple sclerosis, juvenile        rheumatoid arthritis, celiac disease, or alopecia universalis.        -   Especially where X is a self-antigen polypeptide selected            for treating new onset type 1 diabetes mellitus, pediatric            multiple sclerosis or celiac disease.    -   X is a foreign antigen to which patients develop an unwanted        immune response        -   From peanut, including conarachin (Ara h 1)        -   From wheat, including Alpha-gliadin “33-mer” native (SEQ ID            NO:20), Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21),            Alpha-gliadin (SEQ ID NO:22) and Omega-gliadin (SEQ ID            NO:23).        -   From cat, including Fel d 1A (UNIPROT P30438) and Cat            albumin (UNIPROT P49064).        -   From dog, including Can f 1 (UNIPROT 018873) and Dog albumin            (UNIPROT P49822).    -   X is a foreign transplant antigen against which transplant        recipients develop an unwanted immune response, e.g. a human        leukocyte antigen protein.    -   X is an antibody, antibody fragment or ligand that specifically        binds a circulating protein or peptide or antibody, which        circulating protein or peptide or antibody gives rise to        transplant rejection, immune response against a therapeutic        agent, autoimmune disease, and/or allergy.        -   Especially where X binds an endogenous circulating protein            or peptide or antibody.    -   Y is a linker selected from: Formula Ya, Formula Yb, Formula Yh,        Formula Yi, Formula Yk, Formula Ym, Formula Yn, Formula Yo and        Formula Yp.        -   Especially where n is 8 to 90±10%, p is 20 to 100±10%, and q            is 3 to 20±3.            -   Particularly where n is 40 to 80±10%, p is 30 to 40±10%,                and q is 4 to 12±3.        -   Especially where Y is Formula Ya, Formula Yb, Formula Ym or            Formula Yn.            -   Particularly where n is 8 to 90±10%, p is 20 to 100±10%                and q is 3 to 20±3.                -   More particularly where n is 40 to 80±10%, p is 30                    to 40±10%, and q is 4 to 12±3.            -   Particularly where Z is conjugated to Y via an                ethylacetamido group.                -   More particularly where Z is conjugated to Y at its                    C1.                -    More particularly where R⁸ is CMP.                -   More particularly where R⁸ is CMP.            -   Particularly where R⁸ is CMP.    -   Y is a linker selected from: Formula Yc, Formula Yf, Formula Yg        and Formula Ym.        -   Especially where W_(p) is a random copolymer in which R⁹ is            Et-PEG_(t)-AcN and R¹⁰ is 2-hydroxypropyl.            -   Particularly where t is 1 or 2                -   More particularly where t is 1.            -   Particularly where p is about 90 and includes about 30                W¹ and 60 W² comonomers.    -   Z is galactose, galactosamine, N-acetylgalactosamine, glucose,        glucosamine or N-acetylglucosamine.        -   Especially where Z is galactose or N-acetylgalactosamine            conjugated at C1, C2 or C6.            -   Particularly where Z is galactose or                N-acetylgalactosamine conjugated at C1 or C2.                -   More particularly where Z is N-acetylgalactosamine                    conjugated at C1.        -   Especially where Z is glucose or N-acetylglucosamine            conjugated at C1, C2 or C6.            -   Particularly where Z is glucose or N-acetylglucosamine                conjugated at C1 or C2.                -   More particularly where Z is N-acetylglucosamine                    conjugated at C1.

Each of the above-described groups and sub-groups are individuallypreferred and can be combined to describe further preferred aspects ofthe disclosure, for example but not by way of limitation, as follows:

-   -   X is a self-antigen polypeptide selected for treating type 1        diabetes mellitus, pediatric multiple sclerosis, juvenile        rheumatoid arthritis, celiac disease, or alopecia universalis.        -   Especially where X is a self-antigen polypeptide selected            for treating new onset type 1 diabetes mellitus, pediatric            multiple sclerosis or celiac disease.            -   Particularly where Y is a linker selected from: Formula                Ya, Formula Yb, Formula Yc, Formula Yf, Formula Yg,                Formula Yh, Formula Yi, Formula Yk, Formula Ym, Formula                Yn, Formula Yo and Formula Yp.                -   Especially where W_(p) is a W¹ polymer in which R⁹                    is Et-PEG_(t)-AcN or a random copolymer in which R⁹                    is Et-PEG_(t)-AcN and R¹⁰ is 2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 2.                -    More particularly where t is 1.                -    Particularly where p is about 90                -    More particularly where W_(p) is a random copolymer                    and includes about 30 W¹ and 60 W² comonomers.                -   Especially where n is 8 to 90±10%, p is 20 to                    100±10%, and q is 3 to 20±3.                -    Particularly where n is 40 to 80±10%, p is 30 to                    40±10%, and q is 4 to 12±3.                -   Especially where Y is Formula Ya, Formula Yb,                    Formula Ym or Formula Yn.                -    Particularly where n is 8 to 90±10%, p is 20 to                    100±10% and q is 3 to 20±3.                -    More particularly where n is 40 to 80±10%, p is 30                    to 40±10%, and q is 4 to 12±3.                -    Even more particularly where Z is conjugated to Y                    via an ethylacetamido group.                -    More particularly where Z is conjugated to Y via an                    ethylacetamido group.                -    Particularly where Z is conjugated to Y via an                    ethylacetamido group.                -   Especially where Z is galactose, galactosamine or                    N-acetylgalactosamine.                -    Particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1, C2 or C6.                -    More particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1 or C2.                -    P Even more particularly where Z is                    N-acetylgalactosamine conjugated at C1.                -   Especially where Z is glucose, glucosamine or                    N-acetylglucosamine.                -    Particularly where Z is glucose or                    N-acetylglucosamine conjugated at C1, C2 or C6.                -    More particularly where Z is glucose or                    N-acetylglucosamine conjugated at C1 or C2.                -    Even more particularly where Z is                    N-acetylglucosamine conjugated at C1.            -   Particularly where Y is a linker selected from: Formula                Yc, Formula Yf, Formula Yg and Formula Ym.                -   Especially where W_(p) is a random copolymer in                    which R⁹ is Et-PEG_(t)-AcN and R¹⁰ is                    2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 1.                -    Particularly where p is about 90 and includes about                    30 W¹ and 60 W² comonomers.            -   Particularly where Y is a linker selected from: Formula                Yc and Formula Ym.                -   Especially where W_(p) is a random copolymer in                    which R⁹ is Et-PEG_(t)-AcN and R¹⁰ is                    2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 1.                -    Particularly where p is about 90 and includes about                    30 W¹ and 60 W² comonomers.            -   Particularly where Z is galactose, galactosamine or                N-acetylgalactosamine.                -   Especially where Z is galactose or                    N-acetylgalactosamine conjugated at C1, C2 or C6.                -    Particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1 or C2.                -    More particularly where Z is N-acetylgalactosamine                    conjugated at C1.            -   Particularly where Z is glucose, glucosamine or                N-acetylglucosamine.                -   Especially where Z is glucose or N-acetylglucosamine                    conjugated at C1, C2 or C6.                -    More particularly where Z is glucose or                    N-acetylglucosamine conjugated at C1 or C2.                -    Even more particularly where Z is                    N-acetylglucosamine conjugated at C1.        -   Especially where Y is a linker selected from: Formula Ya,            Formula Yb, Formula Yh, Formula Yi, Formula Yk, Formula Ym,            Formula Yn, Formula Yo and Formula Yp.            -   Particularly where Y is a linker selected from: Formula                Yc, Formula Yf, Formula Yg and Formula Ym.                -   Especially where W_(p) is a random copolymer in                    which R⁹ is Et-PEG_(t)-AcN and R¹⁰ is                    2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 1.                -    Particularly where p is about 90 and includes about                    30 W¹ and 60 W² comonomers.            -   Particularly where Y is a linker selected from: Formula                Yc and Formula Ym.                -   Especially where W_(p) is a random copolymer in                    which R⁹ is Et-PEG_(t)-AcN and R¹⁰ is                    2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 1.                -    Particularly where p is about 90 and includes about                    30 Wand 60 W² comonomers.            -   Particularly where n is 8 to 90±10%, p is 20 to 100±10%,                and q is 3 to 20±3.                -   More particularly where n is 40 to 80±10%, p is 30                    to 40±10%, and q is 4 to 12±3.            -   Particularly where Y is Formula Ya, Formula Yb, Formula                Ym or Formula Yn.                -   More particularly where n is 8 to 90±10%, p is 20 to                    100±10% and q is 3 to 20±3.                -    More preferably where n is 40 to 80±10%, p is 30 to                    40±10%, and q is 4 to 12±3.                -   More particularly where Z is conjugated to Y via an                    ethylacetamido group.        -   Especially where Z is galactose, galactosamine or            N-acetylgalactosamine.            -   Particularly where Z is galactose or                N-acetylgalactosamine conjugated at C1, C2 or C6.                -   More particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1 or C2.                -    More preferably where Z is N-acetylgalactosamine                    conjugated at C1.                -   More particularly where Y is a linker selected from:                    Formula Yc, Formula Yf, Formula Yg and Formula Ym.                -    Especially where W_(p) is a random copolymer in                    which R⁹ is Et-PEG_(t)-AcN and R¹⁰ is                    2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 1.                -    Particularly where p is about 90 and includes about                    30 W¹ and 60 W² comonomers.        -   Especially where Z is glucose, glucosamine or            N-acetylglucosamine.            -   Particularly where Z is glucose or N-acetylglucosamine                conjugated at C1, C2 or C6.                -   More particularly where Z is glucose or                    N-acetylglucosamine conjugated at C1 or C2.                -    More preferably where Z is N-acetylglucosamine                    conjugated at C1.                -   More particularly where Y is a linker selected from:                    Formula Yc, Formula Yf, Formula Yg and Formula Ym.                -    Especially where W_(p) is a random copolymer in                    which R⁹ is Et-PEG_(t)-AcN and R¹⁰ is                    2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 1.                -    Particularly where p is about 90 and includes about                    30 W¹ and 60 W² comonomers.                -   More particularly where Y is a linker selected from:                    Formula Yc and Formula Ym.                -    Especially where W_(p) is a random copolymer in                    which R⁹ is Et-PEG_(t)-AcN and R¹⁰ is                    2-hydroxypropyl.                -    Particularly where t is 1 or 2                -    More particularly where t is 1.                -    Particularly where p is about 90 and includes about                    30 W¹ and 60 W² comonomers.    -   m is an integer from about 1 to 100.        -   m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,            17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75,            80, 85, 90, 95, 100 or 110.        -   Particularly m is from about 1 to 20.            -   m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,                16, 17, 18, 19, 20, 21 or 22.            -   More particularly m is about 10.                -   m is 9, 10 or 11.    -   n is an integer representing a mixture including from about 1 to        100        -   n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,            17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50, 54,            55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100,            105 or 110.            -   Particularly n is about 8 to 90.            -   Particularly n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,                18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50, 54,                55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95 or                99.                -   More particularly n is about 40 to 80.                -   More particularly n is 37, 40, 41, 45, 50, 54, 55,                    59, 60, 65, 70, 75, 80, 82, 83 or 88.        -   n represents a mixture encompassing the ranges 1-4, 2-4,            2-6, 3-8, 7-13, 6-14, 15-25, 26-30, 42-50, 46-57, 60-82,            85-90, 90-110 and 107-113.            -   Particularly n represents a mixture encompassing the                ranges 7-13, 6-14, 15-25, 26-30, 42-50, 46-57, 60-82,                85-90 and 82-99.                -   More particularly n represents a mixture                    encompassing the ranges 36-44, 42-50, 46-57, 60-82                    and 75-85.        -   p is an integer representing a mixture including from about            2 to 150.            -   p is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,                17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70,                75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 or                165.            -   Particularly where n is an integer representing a                mixture including from about 1 to 100.                -   Particularly n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,                    12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34,                    35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75,                    80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110.                -    More particularly where n is about 8 to 90.                -    More particularly n is 8, 9, 10, 11, 12, 13, 14,                    15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,                    41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83,                    85, 88, 90, 95 or 99.                -    Even more particularly where n is about 40 to 80.                -    Even more particularly n is 37, 40, 41, 45, 50, 54,                    55, 59, 60, 65, 70, 75, 80, 82, 83 or 88.                -   More particularly p is 18, 19, 20, 25, 30, 35, 40,                    45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100 or                    110.                -    Particularly where n is an integer representing a                    mixture including from about 1 to 100.                -    Particularly n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,                    11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30,                    34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70,                    75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110.                -    More particularly where n is about 8 to 90.                -    More particularly n is 8, 9, 10, 11, 12, 13, 14,                    15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,                    41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83,                    85, 88, 90, 95 or 99.                -    Even more particularly where n is about 40 to 80.                -    Even more particularly n is 37, 40, 41, 45, 50, 54,                    55, 59, 60, 65, 70, 75, 80, 82, 83 or 88.                -    More particularly p is 27, 28, 29, 30, 31, 32, 33,                    34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.                -    Particularly where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,                    10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25,                    30, 34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65,                    70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or                    110.                -    More particularly where n is about 8 to 90.                -    More particularly n is 8, 9, 10, 11, 12, 13, 14,                    15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,                    41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83,                    85, 88, 90, 95 or 99.                -    Even more particularly where n is about 40 to 80.                -    Even more particularly n is 37, 40, 41, 45, 50, 54,                    55, 59, 60, 65, 70, 75, 80, 82, 83 or 88.        -   q is an integer representing a mixture including from about            1 to 44.            -   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,                16, 17, 18, 19, 20, 25, 30, 35, 40, 44 or 48.

Utility, Testing and Administration

General Utility

The compositions of the disclosure find use in a variety of applicationsincluding, as will be appreciated by those in the art, treatment oftransplant rejection, immune response against a therapeutic agent,autoimmune disease, and food allergy, among other uses.

In a preferred embodiment, the compositions of the disclosure are usedto modulate, particularly down-regulate, antigen-specific undesirableimmune response.

The compositions of the disclosure are useful, in additionalembodiments, to bind and clear from the circulation specific undesiredproteins, including antibodies endogenously generated in a patient(i.e., not exogenous antibodies administered to a patient), peptides andthe like, which cause autoimmunity and associated pathologies, allergy,inflammatory immune responses, and anaphylaxis.

In several embodiments according to the present disclosure, antigens aretargeted to the liver for presentation via antigen-presenting cells tospecifically down-regulate the immune system or for clearance ofunwanted circulating proteins. This is distinct from previous uses ofliver targeting, for example as described in US 2013/0078216, where thepurpose of liver-targeting molecules such as DOM26h-196-61 was thedelivery of therapeutic agents to treat liver diseases such as fibrosis,hepatitis, Cirrhosis and liver cancer.

According to several embodiments, the present disclosure providescompositions and methods to treat unwanted immune response toself-antigens and foreign antigens, including but not limited to: aforeign transplant antigen against which transplant recipients developan unwanted immune response (e.g., transplant rejection), a foreignantigen to which patients develop an unwanted immune (e.g., allergic orhypersensitivity) response, a therapeutic agent to which patientsdevelop an unwanted immune response (e.g., hypersensitivity and/orreduced therapeutic activity), a self-antigen to which patients developan unwanted immune response (e.g., autoimmune disease)

Autoimmune disease states that can be treated using the methods andcompositions provided herein include, but are not limited to: AcuteDisseminated Encephalomyelitis (ADEM); Acute interstitial allergicnephritis (drug allergies); Acute necrotizing hemorrhagicleukoencephalitis; Addison's Disease; Alopecia areata; Alopeciauniversalis; Ankylosing Spondylitis; Arthritis, juvenile; Arthritis,psoriatic; Arthritis, rheumatoid; Atopic Dermatitis; Autoimmune aplasticanemia; Autoimmune gastritis; Autoimmune hepatitis; Autoimmunehypophysitis; Autoimmune oophoritis; Autoimmune orchitis; Autoimmunepolyendocrine syndrome type 1; Autoimmune polyendocrine syndrome type 2;Autoimmune thyroiditis; Behcet's disease; Bronchiolitis obliterans;Bullous pemphigoid; Celiac disease; Churg-Strauss syndrome; Chronicinflammatory demyelinating polyneuropathy; Cicatricial pemphigoid;Crohn's disease; Coxsackie myocarditis; Dermatitis herpetiformisDuhring; Diabetes mellitus (Type 1); Erythema nodosum; Epidermolysisbullosa acquisita, Giant cell arteritis (temporal arteritis); Giant cellmyocarditis; Goodpasture's syndrome; Graves' disease; Guillain-Barresyndrome; Hashimoto's encephalitis; Hashimoto's thyroiditis;IgG4-related sclerosing disease; Lambert-Eaton syndrome; Mixedconnective tissue disease; Mucha-Habermann disease; Multiple sclerosis;Myasthenia gravis; Optic neuritis; Neuromyelitis optica; Pemphigusvulgaris and variants; Pernicious angemis; Pituitary autoimmune disease;Polymyositis; Postpericardiotomy syndrome; Premature ovarian failure;Primary Biliary Cirrhosis; Primary sclerosing cholangitis; Psoriasis;Rheumatic heart disease; Sjogren's syndrome; Systemic lupuserythematosus; Systemic sclerosis; Ulcerative colitis; Undifferentiatedconnective tissue disease (UCTD); Uveitis; Vitiligo; and Wegener'sgranulomatosis.

A particular group of autoimmune disease states that can be treatedusing the methods and compositions provided herein include, but are notlimited to: Acute necrotizing hemorrhagic leukoencephalitis; Addison'sDisease; Arthritis, psoriatic; Arthritis, rheumatoid; Autoimmuneaplastic anemia; Autoimmune hypophysitis; Autoimmune gastritis;Autoimmune polyendocrine syndrome type 1; Bullous pemphigoid; Celiacdisease; Coxsackie myocarditis; Dermatitis herpetiformis Duhring;Diabetes mellitus (Type 1); Epidermolysis bullosa acquisita; Giant cellmyocarditis; Goodpasture's syndrome; Graves' disease; Hashimoto'sthyroiditis; Mixed connective tissue disease; Multiple sclerosis;Myasthenia gravis; Neuromyelitis optica; Pernicious angemis; Pemphigusvulgaris and variants; Pituitary autoimmune disease; Premature ovarianfailure; Rheumatic heart disease; Systemic sclerosis; Sjogren'ssyndrome; Systemic lupus erythematosus; and Vitiligo.

In the embodiments employing an antigen against which an unwanted immuneresponse is developed, such as food antigens, treatment can be providedfor reactions against, for example: peanut, apple, milk, egg whites, eggyolks, mustard, celery, shrimp, wheat (and other cereals), strawberryand banana.

As will be appreciated by those skilled in the art, a patient can betested to identify a foreign antigen against which an unwanted immuneresponse has developed, and a composition of the disclosure can bedeveloped based on that antigen.

Testing

In establishing the utility of the compositions and methods of thedisclosure, specificity in binding to antigen-presenting cells in theliver (particularly binding to hepatocytes and specifically ASGPR)should initially be determined. This can be accomplished, for example,by employing a marker (such as the fluorescent marker phycoerythrin(“PE”)) in a composition of the disclosure. The composition isadministered to suitable experimental subjects. Controls, e.g.,unconjugated PE or vehicle (saline) are administered to other group(s)of subjects. The composition and controls are allowed to circulate for aperiod of 1 to 5 hours, after which the spleens and livers of thesubjects are harvested and measured for fluorescence. The specific cellsin which fluorescence is found can be subsequently identified.Compositions of the disclosure, when tested in this manner, show higherlevels of concentration in the antigen-presenting cells of the liver ascompared with unconjugated PE or vehicle.

Effectiveness in immune modulation can be tested by measuring theproliferation of OT-I CD8+ cells (transplanted into host mice) inresponse to the administration of a composition of the disclosureincorporating a known antigen, such as ovalbumin (“OVA”), as comparedwith administration of the antigen alone or just vehicle. Compositionsof the disclosure, when tested in this manner, show an increase of OT-Icell proliferation as compared with antigen alone or vehicle,demonstrating increased CD8+ T-cell cross-priming. To distinguish Tcells being expanded into a functional effector phenotype from thosebeing expanded and deleted, the proliferating OT-I CD8⁺ T cells can bephenotypically analyzed for molecular signatures of exhaustion [such asprogrammed death-1 (PD-1), FasL, and others], as well as Annexin-Vbinding as a hallmark of apoptosis and thus deletion. The OT-I CD8⁺ Tcells can also be assessed for their responsiveness to an antigenchallenge with adjuvant in order to demonstrate functionalnon-responsiveness, and thus immune tolerance, towards the antigen. Todo so, the cells are analyzed for inflammatory signatures afteradministration of compositions of the disclosure into host mice followedby an antigen challenge. Compositions of the disclosure when tested inthis manner demonstrate very low (e.g., background) levels ofinflammatory OT-I CD8⁺ T cell responses towards OVA in comparison tocontrol groups, thus demonstrating immune tolerance.

Humoral immune response can be tested by administering a composition ofthe disclosure incorporating a known antigen, such as OVA, as comparedwith the administration of the antigen alone or just vehicle, andmeasuring the levels of resulting antibodies. Compositions of thedisclosure when tested in this manner show very low (e.g., background)levels of antibody formation responsive to their administration and theadministration of vehicle, with significantly higher levels of antibodyformation responsive to administration of the antigen.

Effectiveness in tolerization against an antigen can be tested as abovewith reference to humoral immune response, where several weeks followingtreatment(s) with a composition of the disclosure a group of subjects ischallenged by administration of the antigen alone, followed by measuringthe levels of antibodies to the antigen. Compositions of the disclosurewhen tested in this manner show low levels of antibody formationresponsive to challenge with the antigen in groups pretreated with suchcompositions as compared to groups that are not pretreated.

Disease-focused experimental models are well known to those skilled inthe art and include the NOD (or non-obese diabetic) mouse model ofautoimmunity and tolerance and the EAE (experimental autoimmuneencephalomyelitis) model for the human inflammatory demyelinatingdisease, multiple sclerosis. In particular, the NOD mouse developsspontaneous autoimmune diabetes (similar to type 1a diabetes in humans).Groups of NOD mice are treated with test compound or a negative control,followed by measurement of blood glucose. Successful treatmentcorresponds to likelihood of treating diabetes in humans or proof ofmechanism for approaches to the treatment of other autoimmune diseases.(See, e.g., Anderson and Bluestone, Annu. Rev. Immunol. 2005;23:447-85.)

Administration

The compositions of the disclosure are administered at a therapeuticallyeffective dosage, e.g., a dosage sufficient to provide treatment for thedisease states previously described. Administration of the compounds ofthe disclosure or the pharmaceutically acceptable salts thereof can bevia any of the accepted modes of administration for agents that servesimilar utilities.

While human dosage levels have yet to be optimized for the compounds ofthe disclosure, these can initially be extrapolated from the about 10 μgto 100 μg doses administered for mice. Generally, an individual humandose is from about 0.01 to 2.0 mg/kg of body weight, preferably about0.1 to 1.5 mg/kg of body weight, and most preferably about 0.3 to 1.0mg/kg of body weight. Treatment can be administered for a single day ora period of days, and can be repeated at intervals of several days, oneor several weeks, or one or several months. Administration can be as asingle dose (e.g., as a bolus) or as an initial bolus followed bycontinuous infusion of the remaining portion of a complete dose overtime, e.g., 1 to 7 days. The amount of active compound administeredwill, of course, be dependent on any or all of the following: thesubject and disease state being treated, the severity of the affliction,the manner and schedule of administration and the judgment of theprescribing physician. It will also be appreciated that amountsadministered will depend upon the molecular weight of the antigen,antibody, antibody fragment or ligand as well as the size of the linker.

The compositions of the disclosure can be administered either alone orin combination with other pharmaceutically acceptable excipients. Whileall typical routes of administration are contemplated (e.g. oral,topical, transdermal, injection (intramuscular, intravenous, orintra-arterial)), it is presently preferred to provide liquid dosageforms suitable for injection. The formulations will typically include aconventional pharmaceutical carrier or excipient and a composition ofthe disclosure or a pharmaceutically acceptable salt thereof. Inaddition, these compositions can include other medicinal agents,pharmaceutical agents, carriers, and the like, including, but notlimited to the therapeutic protein, peptide, antibody or antibody-likemolecule corresponding to the antigen (X) employed in the composition ofthe disclosure, and other active agents that can act asimmune-modulating agents and more specifically can have inhibitoryeffects on B-cells, including anti-folates, immune suppressants,cyostatics, mitotic inhibitors, and anti-metabolites, or combinationsthereof.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 95%,preferably about 0.5% to 50%, by weight of a composition of thedisclosure, the remainder being suitable pharmaceutical excipients,carriers, etc. Dosage forms or compositions containing active ingredientin the range of 0.005% to 95% with the balance made up from non-toxiccarrier can be prepared.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active composition of thedisclosure (e.g., a lyophilized powder) and optional pharmaceuticaladjuvants in a carrier, such as, for example, water (water forinjection), saline, aqueous dextrose, glycerol, glycols, ethanol or thelike (excluding galactoses), to thereby form a solution or suspension.If desired, the pharmaceutical composition to be administered can alsocontain minor amounts of nontoxic auxiliary substances such as wettingagents, emulsifying agents, stabilizing agents, solubilizing agents, pHbuffering agents and the like, for example, sodium acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine acetate and triethanolamine oleate, etc., osmolytes,amino acids, sugars and carbohydrates, proteins and polymers, salts,surfactants, chelators and antioxidants, preservatives, and specificligands. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington: The Science and Practice of Pharmacy, Pharmaceutical Press,22nd Edition, 2012. The composition or formulation to be administeredwill, in any event, contain a quantity of the active compound in anamount effective to treat the symptoms of the subject being treated.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe above-described disclosure, as well as to set forth the best modescontemplated for carrying out various aspects of the disclosure. It isunderstood that these examples in no way serve to limit the true scopeof this disclosure, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Example 1 F1aA-OVA-m₄-n₈₀ (or F1a-OVA-m₄-n₈₀-2NGAL)

1A. Formula 103′ where X′ is OVA and m is 4

In an endotoxin-free tube, OVA (5.0 mg, 0.00012 mmol) was added to 100μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg ofTraut's Reagent was dissolved in 100 μl of pH 7.0 PBS, and 16 μl(0.00119 mmol) of the Traut's Reagent solution so obtained was added tothe stirred solution of OVA with continued stirring. After 1 hour,excess Traut's Reagent was removed using a centrifugal size exclusioncolumn to afford the corresponding product of Formula 103′.

1B. Formula 106A where n is 80

In an endotoxin-free tube, galactosamine (10.0 mg, 0.04638 mmol) wasdissolved with stirring in 100 μl of pH 8.0 PBS containing 5 mM EDTA.Pyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104 where n is80) (16.23 mg, 0.00464 mmol) dissolved in 100 μl of pH 7.0 PBS was addedto the stirring solution of galactosamine. After 1 hour, the resultingpyridyl dithiol-poly(ethylene glycol)-N-acetylgalactosamine (Formula106A) was ready to be used without further purification.

1C. Formula 1aA where X′ is OVA, m is 4, n is 80 (and Z′ is C2Galactosamine)

The purified OVA-Traut conjugate of Formula 103′ prepared in Example 1Awas added directly to the stirring product of Formula 106A prepared inExample 1B. After 1 hour, the resulting product of Formula 1a waspurified by passing the reaction mixture through a centrifugal sizeexclusion column. Characterization (UHPLC SEC, gel electrophoresis)confirmed product identity. (See FIG. 5 .)

1D. Other Compounds of Formula 103′

By following the procedure described in Example 1A and substituting OVAwith the following:

-   -   Abciximab,    -   Adalimumab,    -   Agalsidase alfa,    -   Agalsidase beta,    -   Aldeslukin,    -   Alglucosidase alfa,    -   Factor VIII,    -   Factor IX,    -   L-asparaginase,    -   Laronidase,    -   Octreotide,    -   Phenylalanine ammonia-lyase,    -   Rasburicase,    -   Insulin (SEQ ID NO:1),    -   GAD-65 (SEQ ID NO:2),    -   IGRP (SEQ ID NO:3)    -   MBP (SEQ ID NO:4),    -   MOG (SEQ ID NO:5),    -   PLP (SEQ ID NO:6),    -   MBP13-32 (SEQ ID NO:7),    -   MBP83-99 (SEQ ID NO:8),    -   MBP111-129 (SEQ ID NO:9),    -   MBP146-170 (SEQ ID NO:10),    -   MOG1-20 (SEQ ID NO:11),    -   MOG35-55 (SEQ ID NO:12),    -   PLP139-154 (SEQ ID NO:13),    -   MART1 (SEQ ID NO:14),    -   Tyrosinase (SEQ ID NO:15),    -   PMEL (SEQ ID NO:16),    -   Aquaporin-4 (SEQ ID NO:17),    -   S-arrestin (SEQ ID NO:18),    -   IRBP (SEQ ID NO:19),    -   Conarachin (UNIPROT Q6PSU6),    -   Alpha-gliadin “33-mer” native (SEQ ID NO:20),    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21),    -   Alpha-gliadin (SEQ ID NO:22),    -   Omega-gliadin (SEQ ID NO:23),    -   Fel d 1A (UNIPROT P30438),    -   Cat albumin (UNIPROT P49064),    -   Can f 1 (UNIPROT 018873),    -   Dog albumin (UNIPROT P49822), and    -   RhCE (UNIPROT P18577),        there are obtained the following corresponding compounds of        Formula 103′ where:    -   X is Abciximab and m is 10,    -   X is Adalimumab and m is 11,    -   X is Agalsidase alfa and m is 14,    -   X is Agalsidase beta and m is 14,    -   X is Aldeslukin and m is 6,    -   X is Alglucosidase alfa and m is 13,    -   X is Factor VIII and m is 100,    -   X is Factor IX and m is 18,    -   X is L-asparaginase and m is 5,    -   X is Laronidase and m is 7,    -   X is Octreotide and m is 1,    -   X is Phenylalanine ammonia-lyase and m is 12,    -   X is Rasburicase and m is 12,    -   X is Insulin (SEQ ID NO:1) and m is 2,    -   X is GAD-65 (SEQ ID NO:2) and m is 8,    -   X is IGRP (SEQ ID NO:3) and m is 7,    -   X is MBP (SEQ ID NO:4) and m is 6,    -   X is MOG (SEQ ID NO:5) and m is 5,    -   X is PLP (SEQ ID NO:6) and m is 8,    -   X is MBP13-32 (SEQ ID NO:7) and m is 1,    -   X is MBP83-99 (SEQ ID NO:8) and m is 1,    -   X is MBP111-129 (SEQ ID NO:9) and m is 1,    -   X is MBP146-170 (SEQ ID NO:10) and m is 2,    -   X is MOG1-20 (SEQ ID NO:11) and m is 1,    -   X is MOG35-55 (SEQ ID NO:12) and m is 2,    -   X is PLP139-154 (SEQ ID NO:13) and m is 3,    -   X is MART1 (SEQ ID NO:14) and m is 4,    -   X is Tyrosinase (SEQ ID NO:15) and m is 8,    -   X is PMEL (SEQ ID NO:16) and m is 5,    -   X is Aquaporin-4 (SEQ ID NO:17) and m is 4,    -   X is S-arrestin (SEQ ID NO:18) and m is 12,    -   X is IRBP (SEQ ID NO:19) and m is 21,    -   X is Conarachin and m is 21,    -   X is Alpha-gliadin “33-mer” native (SEQ ID NO:20) and m is 1,    -   X is Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21) and m is        1,    -   X is Alpha-gliadin (SEQ ID NO:22) and m is 1,    -   X is Omega-gliadin (SEQ ID NO:23) and m is 1,    -   X is Fel d 1 and m is 4,    -   X is Cat albumin and m is 16,    -   X is Can f 1 and m is 6,    -   X is Dog albumin and m is 23, and    -   X is RhCE and m is 10.

1E. Other Compounds of Formula 1aA

By following the procedure described in Example 1C and substituting thecompounds of Formula 103′, for example as obtained in Example 1D, thereare obtained the following corresponding compounds of Formula 1aA:

-   -   F1aA-Abciximab-m₁₀-n₈₀,    -   F1aA-Adalimumab-m₁₁-n₈₀,    -   F1aA-Agalsidase alfa-m₁₄-n₈₀,    -   F1aA-Agalsidase beta-m₁₄-n₈₀,    -   F1aA-Aldeslukin-m₆-n₈₀,    -   F1aA-Alglucosidase alfa-m₁₃-n₈₀,    -   F1aA-Factor VIII-m₁₀₀-n₈₀,    -   F1aA-Factor IX-m₁₈-n₈₀,    -   F1aA-L-asparaginase-m₅-n₈₀,    -   F1aA-Laronidase-m₇-n₈₀,    -   F1aA-Octreotide-m₁-n₈₀,    -   F1aA-Phenylalanine ammonia-lyase-m₁₂-n₈₀,    -   F1aA-Rasburicase-m₁₂-n₈₀,    -   F1aA-Insulin-m₂-n₈₀,    -   F1aA-GAD-65-m₅-n₈₀,    -   F1aA-IGRP-m₇-n₈₀,    -   F1aA-MBP-m₆-n₈₀,    -   F1aA-MOG-m₅-n₈₀,    -   F1aA-PLP-m₅-n₈₀,    -   F1aA-MBP13-32-m₁-n₈₀,    -   F1aA-MBP83-99-m₁-n₈₀,    -   F1aA-MBP111-129-m₁-n₈₀,    -   F1aA-MBP146-170-m₂-n₈₀,    -   F1aA-MOG1-20-m₁-n₈₀,    -   F1aA-MOG35-55-m₂-n₈₀,    -   F1aA-PLP139-154-m₃-n₈₀,    -   F1aA-MART1-m₄-n₈₀    -   F1aA-Tyrosinase-m₅-n₈₀,    -   F1aA-PMEL-m₅-n₈₀,    -   F1aA-Aquaporin-4-m₄-n₈₀,    -   F1aA-S-arrestin-m₁₂-n₈₀,    -   F1aA-IRBP-m₂₁-n₈₀,    -   F1aA-Conarachin-m₂₁-n₈₀,    -   F1aA-Alpha-gliadin “33-mer” native-m₁-n₈₀,    -   F1aA-Alpha-gliadin “33-mer” deamidated-m₁-n₈₀,    -   F1aA-Alpha-gliadin-m₁-n₈₀,    -   F1aA-Omega-gliadin-m₁-n₈₀,    -   F1aA-Fel d 1-m₄-n₈₀,    -   F1aA-Cat albumin-m₁₆-n₈₀,    -   F1aA-Can f 1-m₆-n₈₀,    -   F1aA-Dog albumin-m₂₃-n₈₀, and    -   F1aA-RhCE-m₁₀-n₈₀.

1F. Other Compounds of Formula 106A

By following the procedure described in Example 1B and substituting thepyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104 where n is80) with the following:

-   -   Formula 104 where n is 12,    -   Formula 104 where n is 33,    -   Formula 104 where n is 40,    -   Formula 104 where n is 43,    -   Formula 104 where n is 50,    -   Formula 104 where n is 60,    -   Formula 104 where n is 75, and    -   Formula 104 where n is 80,        there are obtained the following corresponding compounds of        Formula 106A where:    -   n is 12,    -   n is 33,    -   n is 40,    -   n is 43,    -   n is 50,    -   n is 60,    -   n is 75, and    -   n is 84,

1G. Other Compounds of Formula 1aA

By following the procedure described in Example 1E and substituting thecompound of Formula 106A with the compounds obtained in Example 1F,there are obtained the corresponding compounds of Formula 1aA where n is12, 33, 40, 43, 50, 60, 75 and 84, such as:

-   -   F1aA-Insulin-m₂-n₁₂,    -   F1aA-Insulin-m₂-n₃₃,    -   F1aA-Insulin-m₂-n₈₀,    -   F1aA-Insulin-m₂-n₄₃,    -   F1aA-Insulin-m₂-n₅₀,    -   F1aA-Insulin-m₂-n₆₀,    -   F1aA-Insulin-m₂-n₇₅, and    -   F1aA-Insulin-m₂-n₈₄.

1H. Other Compounds of Formula 1aA

Similarly, by following the procedures described in Example 1A-G andsubstituting the compound glucosamine for galactosamine, there areobtained the corresponding compounds of Formula 1aA where Z′ is C2glucosamine.

Example 2 F1b-OVA-m₁-n₄-p34-2NAcGAL

2A. Formula 103′ where X′ is Ovalbumin and m is 1

In an endotoxin-free tube, OVA (6.5 mg, 0.000155 mmol) was added to 200μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg ofTaut's Reagent was dissolved in 100 μl of pH 7.0 PBS, and 43 μl (0.00310mmol) of the Traut's Reagent solution so obtained was added to thestirred solution of OVA with continued stirring. After 1 hour,non-reacted Traut's Reagent was removed using a centrifugal sizeexclusion column to afford the product of Formula 103′.

2B. Formula 1b where X′ is Ovalbumin, m is 1, n is 4, p is 34, R⁹ is aDirect Bond and Z″ is 2NAcGAL

In a micro centrifuge tube, poly(Galactosamine Methacrylate)-(pyridyldisulfide) (Formula 201) (20.0 mg, 0.0020 mmol) was solubilized in 50 μlof pH 8.0 PBS containing 5 mM EDTA. To this was added the purifiedOVA-Traut product from Example 2A followed by stirring for 1 hour. Theresulting product of Formula 1b was purified by passing the reactionmixture through a centrifugal size exclusion column. Characterization(UHPLC SEC, gel electrophoresis) confirmed the identity of the product.(See FIG. 5 .)

2C. Other Compounds of Formula 1b

By following the procedure described in Example 2B and substituting thecompounds of Formula 103′, for example as obtained in Example 1D, thereare obtained the following corresponding compounds of Formula 1b:

-   -   F1b-Abciximab-m₁₀-n₄-p34-2NAcGAL,    -   F1b-Adalimumab-m₁₁-n₄-p34-2NAcGAL,    -   F1b-Agalsidase alfa-m₁₄-n₄-p34-2NAcGAL,    -   F1b-Agalsidase beta-m₁₄-n₄-p34-2NAcGAL,    -   F1b-Aldeslukin-m₆-n₄-p34-2NAcGAL,    -   F1b-Alglucosidase alfa-m₁₃-n₄-p34-2NAcGAL,    -   F1b-Factor VIII-m₁₀₀-n₄-p34-2NAcGAL,    -   F1b-Factor IX-m₁₈-n₄-p34-2NAcGAL,    -   F1b-L-asparaginase-m₅-n₄-p34-2NAcGAL,    -   F1b-Laronidase-m₇-n₄-p34-2NAcGAL,    -   F1b-Octreotide-m₁-n₄-p34-2NAcGAL,    -   F1b-Phenylalanine ammonia-lyase-m₁₂-n₄-p34-2NAcGAL,    -   F1b-Rasburicase-m₁₂-n₄-p₃₄-2NAcGAL,    -   F1b-Insulin-m₂-n₄-p₃₄-2NAcGAL,    -   F1b-GAD-65-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-IGRP-m₇-n₄-p₃₄-2NAcGAL,    -   F1b-MBP-m₆-n₄-p₃₄-2NAcGAL,    -   F1b-MOG-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-PLP-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-MBP13-32-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-MBP83-99-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-MBP111-129-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-MBP146-170-m₂-n₄-p₃₄-2NAcGAL,    -   F1b-MOG1-20-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-MOG35-55-m₂-n₄-p₃₄-2NAcGAL,    -   F1b-PLP139-154-m₃-n₄-p₃₄-2NAcGAL,    -   F1b-MART1-m₄-n₄-p₃₄-2NAcGAL,    -   F1b-Tyrosinase-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-PMEL-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-Aquaporin-4-m₄-n₄-p₃₄-2NAcGAL,    -   F1b-S-arrestin-m₁₂-n₄-p₃₄-2NAcGAL,    -   F1b-IRBP-m₂₁-n₄-p₃₄-2NAcGAL,    -   F1b-Conarachin-m₂₁-n₄-p₃₄-2NAcGAL,    -   F1b-Alpha-gliadin “33-mer” native-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Alpha-gliadin “33-mer” deamidated-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Alpha-gliadin-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Omega-gliadin-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Fel d 1-m₄-n₄-p₃₄-2NAcGAL,    -   F1b-Cat albumin-m₁₆-n₄-p₃₄-2NAcGAL,    -   F1b-Can f 1-m₆-n₄-p₃₄-2NAcGAL,    -   F1b-Dog albumin-m₂₃-n₄-p₃₄-2NAcGAL, and    -   F1b-RhCE-m₁₀-n₄-p₃₄-2NAcGAL.        1D. Other Compounds of Formula 1b

Similarly, by following the procedures described in Example 2B-C andsubstituting the compound poly(Glucosamine Methacrylate)-(pyridyldisulfide) or poly(Galactosamine Methacrylate)-(pyridyl disulfide),there are obtained the corresponding compounds of Formula 1b where Z″ is2-NAcGLU.

Example 3 F1f-OVA-m₁-n₄-p₃₃-2NAcGAL

3A. Formula 1f where X′ is Ovalbumin and m is 1, n is 4, p is 33, R⁹ isa Direct Bond and Z″ is 2NAcGAL

In an endotoxin-free tube, OVA (4.0 mg, 0.0000952381 mmol) was added to0.1 ml of pH 7.4 PBS and stirred. Separately,poly-(n-Acetylgalactosamine)-p-nitrophenyol carbonate of Formula 601where n is 4 and p is 33 (33.0 mg, 0.002380952 mmol) was added to 100 μlof pH 7.5 PBS and vortexed until dissolved. The two solutions werecombined and the mixture was stirred vigorously for 1 hour. The mixturewas then collected and dialyzed for 3 days against pH 7.4 PBS (30 kDamolecular weight cut off) to afford the product of Formula 1f.

3B. Formula 1f where X′ is Ovalbumin and m is 1, n is 4, p is 33, R⁹ isa Direct Bond and Z″ is 2NAcGLU

Similarly, by following the procedure of Example 3A and substitutingpoly-(n-Acetylglucosamine)-p-nitrophenyol carbonate forpoly-(n-Acetylgalactosamine)-p-nitrophenyol carbonate, there is obtainedthe corresponding compound of Formula 1f where Z″ is 2NAcGLU.

Example 4 F1g-PVA-m₁-p₉₀-2NAcGAL

4A. Formula 1q where X′ is Ovalbumin and m is 1, p is 90, R⁹ is a DirectBond and Z″ is 2NAcGAL

In an endotoxin-free tube, OVA (5.0 mg, 0.000119048 mmol) was added to0.2 ml of pH 7.4 PBS and stirred. To the stirring solution was added 75mg (0.00297619 mmol) of Poly(Galactosamine Methacrylate)-NHS (Formula701) dissolved in 0.4 ml of pH 7.4 PBS. The mixture was allowed to stirfor 2 hours. The mixture was then collected and dialyzed for 3 daysagainst pH 7.4 PBS (30 kDa molecular weight cut off) to afford theproduct of Formula 1g.

4B. Formula 1q where X′ is Ovalbumin and m is 1, p is 90, R⁹ is a directbond and Z″ is 2NAcGLU

Similarly, by following the procedure of Example 4A and substitutingPoly(Glucosamine Methacrylate)-NHS for Poly(GalactosamineMethacrylate)-NHS, there is obtained the corresponding compound ofFormula 1g where Z″ is 2NAcGLU.

Example 5 F1h-OVA-m₂-n₄₅-p₅₅-q₄-2NAcGAL

5A. Formula 802′ where X′ is Ovalbumin, m is 2 and n is 45

In an endotoxin-free tube, OVA (3.0 mg, 0.0000714286 mmol) was added to150 μl of pH 8.0 PBS containing 5 mM EDTA and stirred.Dibenzocyclooctyne-PEG-(p-nitrophenyl carbonate) (Formula 801) (5.265mg, 0.002142857 mmol) dissolved in DMF was added to the OVA solution andstirred for 1 hour. The excess dibenzocyclooctyne-PEG-(p-nitrophenylcarbonate) was removed using a centrifugal size exclusion column toafford the product of Formula 802′.

5B. Formula 1h where X′ is Ovalbumin, m is 2, n is 45, p is 55, q is 4,R⁸ is CH₂, R⁹ is a Direct Bond and Z″ is 2NAcGAL

Poly(Galactosamine Methacrylate)-N3 (Formula 803 where p is 55, q is 4and Z″ is N-acetylgalactosamine) (33 mg, 0.002142857 mmol) was dissolvedin 100 μl of pH 7.4 PBS and added to the product of Example 5A withstirring. After 1 hour, the resulting product of Formula 1h was purifiedby centrifugal size exclusion chromatography.

5C. Formula 1h where X′ is Ovalbumin, m is 2, n is 45, p is 55, q is 4,R⁸ is CH₂, R⁹ is a Direct Bond and Z″ is 2NAcGLU

Similarly, by following the procedure of Example 5B and substitutingPoly(Glucosamine Methacrylate)-NHS for Poly(GalactosamineMethacrylate)-NHS, there is obtained the corresponding compound ofFormula 1h where Z″ is 2NAcGLU.

Example 6 F1j-OVA-m₁₀-n₄₅-p₅₅-q₄-2NAcGAL

6A. Formula 103′ where X′ is Ovalbumin and m is 10

In an endotoxin-free tube, OVA (5.0 mg, 0.00019 mmol) was added to 150μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg ofTaut's Reagent was dissolved in 100 μl of pH 7.0 PBS, and 16 μl (0.0019mmol) of the Traut's Reagent solution so obtained was added to thestirred solution of OVA with continued stirring. After 1 hour,non-reacted Traut's Reagent was removed using a centrifugal sizeexclusion column to afford the product of Formula 103′.

6B. Formula 902″ where X′ is Ovalbumin, m is 10 and n is 45

Dibenzocyclooctyne-PEG-(pyridyl disulfide) (Formula 901 where n is 45)(6.0 mg, 0.00238 mmol) was dissolved in DMF and the resulting solutionwas added to the OVA solution obtained in Example 6A and stirred for 1hour. The excess dibenzocyclooctyne-PEG-(pyridyl disulfide) was removedusing centrifugal size exclusion chromatography to afford the product ofFormula 902″.

6C. Formula 1i where X′ is Ovalbumin, m is 10, n is 45, p is 55, q is 4,R⁸ is CH₂, R⁹ is a Direct Bond and Z″ is 2NAcGAL

Poly(Galactosamine Methacrylate)-N3 (Formula 803 where p is 55, q is 4and Z″ is N-acetylgalactosamine) (36 mg, 0.00238 mmol) was dissolved in150 μl of pH 7.4 PBS and added to the product of Example 6B withstirring. After 1 hour, the resulting product of Formula 1j was purified(excess p(GMA)-N3 removed) by centrifugal size exclusion chromatography.Characterization (UHPLC SEC, gel electrophoresis) confirmed the identityof the product.

6D. Formula 1i where X′ is Ovalbumin, m is 10, n is 45, p is 55, q is 4,R⁸ is CH₂, R⁹ is a Direct Bond and Z″ is 2NAcGLU

Similarly, by following the procedure of Example 6C and substitutingPoly(Glucosamine Methacrylate)-NHS for Poly(GalactosamineMethacrylate)-NHS, there is obtained the corresponding compound ofFormula 1j where Z″ is 2NAcGLU.

Example 7 F1L-OVA-m₂-n₈₀-p₅₅-q₄-2NAcGAL

7A. Formula 1002 where X′ is Ovalbumin, m is 2 and n is 80

Dibenzocyclooctyne-PEG-(pyridyl disulfide) (Formula 1001 where n is 80)(9.0 mg, 0.00238 mmol) was dissolved in DMF and the resulting solutionwas added to a purified OVA solution of Formula 103′ (where X′ isOvalbumin and m is 2), for example prepared as described in Example 6Aand stirred for 1 hour. The excess dibenzocyclooctyne-PEG-(pyridyldisulfide) was removed using centrifugal size exclusion chromatographyto afford the product of Formula 1002.

7B. Formula 1L where X′ is Ovalbumin, m is 2, n is 80, p is 55, q is 4,R⁸ is CH₂, R⁹ is a Direct Bond and Z″ is 2NAcGAL

Poly(Galactosamine Methacrylate)-N3 (Formula 803 where p is 55, q is 4and Z″ is N-Acetylgalactosamine) (36 mg, 0.00238 mmol) was dissolved in150 μl of pH 7.4 PBS and added to the product of Example 7A withstirring. After 1 hour, the resulting product of Formula 1L was purified(excess poly(Galactosamine Methacrylate)-N3 removed) by centrifugal sizeexclusion chromatography. Characterization (UHPLC SEC, gelelectrophoresis) confirmed the identity of the product.

7C. Formula 1L where X′ is Ovalbumin, m is 2, n is 80, p is 55, q is 4,R⁸ is CH₂, R⁹ is a Direct Bond and Z″ is 2NAcGLU

Similarly, by following the procedure of Example 7B and substitutingPoly(Glucosamine Methacrylate)-NHS for Poly(GalactosamineMethacrylate)-NHS, there is obtained the corresponding compound ofFormula 1jL where Z″ is 2NAcGLU.

Example 8 Preparation of Poly(Galactosamine Methacrylate) Polymers

8A. Galactosamine Methacrylate

To stirred galactosamine hydrochloride (2.15 g, 10.0 mmol) was added 0.5M sodium methoxide (22 ml, 11.0 mmol). After 30 minutes, methacrylateanhydride (14.694 g, 11.0 mmol) was added and stirring continued for 4hours. The resulting galactosamine methacrylate was loaded onto silicagel via rotovap and purified via column chromatography using DCM:MeOH(85:15).

8B. Formula 201 where n is 4 and p is 30

Galactose methacrylate (600 mg, 2.43 mmol),2-(2-(2-(2-(pyridin-2-yldisulfanyl)ethoxy)ethoxy)ethoxy)ethyl2-((phenylcarbonothioyl)thio)acetate (44.8 mg, 0.081 mmol) and AIBN(3.174089069 mg, 0.016 mmol) were added to 1.5 ml of DMF in a SchlenkFlask. The reaction mixture was subjected to 4 freeze-thaw cycles andthen stirred at 70° C. for 6 hours. The desired polymer product ofFormula 201 was precipitated in 12 ml of methanol, and excess solventwas removed under reduced pressure.

8C. Formula 201 where n is 4 and p is 30

Similarly, by following the procedure of Example 8B and substitutingGlucose methacrylate for galactose methacrylate there are obtained thecorresponding poly(Glucosamine methacrylate) polymers.

Example 9 Preparation of F1aA-PE-m₃-n₈₀

9A. Formula 103′ where X′ is Phycoerythrin

In an endotoxin-free tube, phycoerythrin (“PE”) (purchased from Pierce)(200 μl, 0.000004 mmol) was added to 50 μl of pH 8.0 PBS containing 5 mMEDTA and stirred. Separately, 1 mg of Taut's Reagent was dissolved in100 μl of pH 7.0 PBS, and 2 μl (0.00013 mmol) of the Traut's Reagentsolution so obtained was added to the stirred solution of PE withcontinued stirring. After 1 hour, excess Traut's Reagent was removedusing a centrifugal size exclusion column to afford the product ofFormula 103′

9B. Formula 106A where n is 80

In an endotoxin-free tube, galactosamine (7.0 mg, 0.03246 mmol) wasdissolved with stirring in 100 μl of pH 8.0 PBS containing 5 mM EDTA.Pyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104 where n is80) (16.23 mg, 0.00464 mmol) dissolved in 50 μl of pH 7.0 PBS was addedto the stirring solution of galactosamine. After 1 hour, the resultingproduct of Formula 106A was ready to be used without furtherpurification.

9C. Formula 1a where X′ is Phycoerythrin, m is 3, n is 80 and Z′ isgalactosamine

The purified PE-Traut conjugates prepared in Example 9A were addeddirectly to the stirring product of Formula 106A prepared in Example 9B.After 1 hour, the resulting product of Formula 1a was purified bypassing the reaction mixture through a centrifugal size exclusioncolumn. Characterization (UHPLC SEC, gel electrophoresis) confirmed theidentity of the product.

9D. Formula 1a where X′ is Phycoerythrin, m is 3, n is 80 and Z′ isGlucosamine

Similarly, by following the procedure of Example 9B and C andsubstituting glucosamine for galactosamine there is obtained thecorresponding compound of Formula 1a where Z″ is glucosamine.

Example 10 Hepatic Distribution

10A. F1aA-PE-m₃-n₈₀ was prepared, for example, as described in Example9. A 30 μg/100 μl solution in sterile saline was prepared for injection.

The F1aA-PE-m₃-n₈₀ solution (30 μg) was administered to one of threegroups of C57 black 6 mice 3 per group) via tail vein injection. The twoother groups of mice received an equivalent volume of phycoerythrin in100 μl of saline or saline vehicle. Three hours after administration,the livers and spleens of these animals were harvested and the level ofcellular fluorescents in these organs was determined by flow cytometryas an indication of cellular PE content.

As shown in FIGS. 1A-1D, sinusoidal endothelial cells (LSECs) (1A),hepatocytes (1C), Kupffer cells (KC) (16), and other antigen-presentingcells (APCs) (1D) from the livers of mice treated with F1aA-PE-m₃-n₈₀exhibited at least a three-fold increase in fluorescence as comparedwith animals that received PE solution. No detectable difference influorescence was found in spleen cells harvested from the three groups.These results confirm that F1aA-PE-m₃-n₈₀ has sufficient specificity forbinding to antigen-presenting cells in the liver.

1013. By following the procedure described in Example 10A andsubstituting F1aA-PE-m₃-n₈₀ with the compounds F1b-PE-m₃-n₄-p₃₄-2NAcGAL,F1f-PE-m₃-n₄-p₃₃-2NAcGAL, F1g-PE-m₃-p₉₀-2NAcGAL,F1h-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL, F1j-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL,F1L-PE-m₃-n₈₀-p₅₅-q₄-2NAcGAL, F1m-PE-m₃-n₈₀-p₃₀-q₄-CMP-2NHAc,F1m-PE-m₃-n₆₂-p₃₀-q₈-CMP-2OH, F1n-PE-m₃-n₁-p₃₀-q₄-CMP-2NHAc andF1n-PE-m₃-n₃₃-p₃₀-q₈-CMP-2OH, prepared, for example, as described withreference to Example 9 by substitution for X in Examples 2B, 3, 4, 5B,6B, 7B, 15G, 15L, 16B and 16F, respectively it is confirmed that thecompounds F1aA-PE-m₃-n₈₀ with the compounds F1b-PE-m₃-n₄-p₃₄-2NAcGAL,F1f-PE-m₃-n₄-p₃₃-2NAcGAL, F1g-PE-m₃-p₉₀-2NAcGAL,F1h-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL, F1 j-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL,F1L-PE-m₃-n₈₀-p₅₅-q₄-2NAcGAL, F1m-PE-m₃-n₈₀-p₃₀-q₄-CMP-2NHAc,F1m-PE-m₃-n₆₂-p₃₀-q₈-CMP-2OH, F1n-PE-m₃-n₁-p₃₀-q₄-CMP-2NHAc andFln-PE-m₃-n₃₃-p₃₀-q₈-CMP-2OH have sufficient specificity for binding toantigen-presenting cells in the liver.

10C. By following the procedure described in Example 10A and 106 andsubstituting the corresponding glucosylated compounds for thegalactosylated compounds, it is confirmed that the glucolsylatedcompounds have sufficient specificity for binding to antigen-presentingcells in the liver.

Example 11 Proliferation of Antigen-Specific OT1 CD8+ T Cells

11A. F1aA-OVA-m₄-n₈₀ synthesized, for example, as described in Example1, was prepared as a 10 μg/100 μl saline solution for injection. On day0, 106 OT-1 T cells were fluorescently labeled and adoptivelytransferred into 3 groups of CD 45.2 mice (5 per group) via tail veininjection. The next day (i.e. Day 1), to each of the 3 groups of micewere administered, respectively, 10 μg of F1aA-OVA-m4-n₈₀, OVA or salinevia tail vein injection. On day 6, the animals were sacrificed and the %of splenic proliferating OT-1 cells was determined via florescenceactivated cell sorting.

The results from this study (see FIG. 2 ) show that the percentage ofproliferating OTI T cells in mice treated with F1aA-OVA-m₄-n₈₀(“Gal-OVA” in FIG. 2 ) was significantly greater than the percentage ofproliferating OTI cells in the spleens of mice treated with OVA orsaline (“nave” in FIG. 2 ). The increase in OTI cell-proliferationdemonstrates the increased CD8+ T-cell cross-priming in animals treatedwith F1aA-OVA-m₄-n₈₀ versus the other therapies. In concert with theresults from Example 12, these results indicate that the ability ofF1aA-OVA-m₄-n₈₀ to target antigens to the liver increases OVApresentation by antigen presenting cells in the liver to OVA-specificOTI T cells.

11B. To distinguish T cells being expanded into a functional effectorphenotype from those being expanded and deleted, the proliferating OTICD8⁺ T cells were analyzed for phosphatidylserine exposure by way ofAnnexin-V binding, as a hallmark of apoptosis and thus deletion, as wellas the exhaustion marker programmed death-1 (PD-1). As shown in FIGS.3A-3B, F1aA-OVA-m₄-n₈₀ (“Gal-OVA” in FIGS. 3A-3B) induced much highernumbers of Annexin-V⁺ and PD-1⁺ proliferating OTI CD8⁺ T cells thansoluble OVA. These data demonstrate that, in accordance with severalembodiments disclosed herein, coupling an antigen to which tolerance isto be induced with linkers and liver targeting moieties as disclosedherein result in unexpectedly enhanced generation of T cells having thecapacity to be immunologically functional.

11C. By following the procedure described in Examples 11A and 11B, andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formula 1 obtained,for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it isshown the compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G induce muchhigher numbers of Annexin-V⁺ and PD-1⁺ proliferating OTI CD8⁺ T cellsthan soluble OVA.

11D. By following the procedure described in Examples 11A and 11B andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formulae 1 and 2obtained, for example, as described in Examples 1E, 1G, 2C, 15I, 15L,16B, 16D and 16F, and substituting OVA with the antigens correspondingto X (or X′ or X″), respectively, it is shown that the compounds fromExamples 1E, 1G, 2C, 15I, 15L, 16B, 16D and 16F induce much highernumbers of Annexin-V⁺ and PD-1⁺ proliferating OTI CD8⁺ T cells thansoluble antigen X.

11E. By following the procedure described in Example 11A-D andsubstituting the corresponding glucosylated compounds for thegalactosylated compounds, it is confirmed that the glucolsylatedcompounds induce much higher numbers of Annexin-V⁺ and PD-1⁺proliferating OTI CD8⁺ T cells than soluble antigen X.

Example 12 F1aA-OVA-m₄-n₈ does not Induce an OVA-Specific AntibodyResponse

12A. In order to assess the humoral immune response to F1aA-OVA-m₄-n₈ wetreated mice with a weekly i.v. injection of either F1aA-OVA-m₄-n₈ orOVA, then measured the levels of OVA-specific antibodies in the blood.On day 0, 7, and 14 of the experiment, mice were administered an i.v.injection of 100 μl of saline containing one of the following: 1.) 6 μgof OVA; 2.) 6 μg of F1aA-OVA-m₄-n₈; 3.) 30 μg of OVA; 4.) 30 μg ofF1aA-OVA-m₄-n₈, or 5.) saline alone. Each group contained 5 mice. On day19, the mice were bled via cheek puncture, and the titer of OVA-specificantibodies in each mouse's blood was determined via ELISA. The resultsfor this study show that although mice treated with 6 and 30 μg of OVAhad increased OVA-specific antibody titers, mice treated with both 6 and30 μg of F1aA-OVA-m₄-n₈ (“Gal-OVA” in FIG. 4 ) had blood titers similarto mice treated with saline (i.e. vehicle treated animals) (FIG. 4 ).For example mice treated with 6 and 30 μg of OVA had an average antibodytiter of 3.5 and 2.5, respectively; whereas, mice treated with 6 and 30μg of OVA had an average antibody titer of 0.75 and 0.25, respectively.Thus, these data demonstrate that coupling an antigen to which immunetolerance is desired to a linker and liver targeting moiety according toseveral embodiments disclosed herein results in significantly lessantigen specific antibody generation. As such, these data demonstratethat the immune response to the antigen delivered to the liver by thecompositions disclosed herein is reduced.

12B. By following the procedure described in Example 12A andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formula 1 obtained,for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 15G, it isshown that mice treated with the compounds from Examples 3A, 4A, 5B, 6C,7B and 15G have OVA-specific antibody titers similar to mice treatedwith saline.

12C. By following the procedure described in Example 12B andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formula 1 obtained,for example, as described in Examples 1E, 1G, 2C, 15I, 15L, 16B, 16D and16F, and substituting OVA with the antigens corresponding to X (or X′ orX″), respectively, it is shown that mice treated with the compounds fromExamples 1E, 1G, 2C, 15I, 15L, 16B, 16D and 16F have antigen X-specificantibody titers similar to mice treated with saline.

12D. By following the procedure described in Example 12A-C andsubstituting the corresponding glucosylated compounds for thegalactosylated compounds, it is confirmed that the glucolsylatedcompounds have antigen X-specific antibody titers similar to micetreated with saline.

Example 13 F1aA-OVA-m₄-n₈ Depletes OVA-Specific Antibodies

13A. Mice that had different OVA-antibody blood titers (each mouse had atiter from 0 to 4.5) were treated with an i.v. injection of 20 μg ofF1aA-OVA-m₄-n₈ solubilized in 100 μl saline. Mice were given i.v.injections of F1aA-OVA-m₄-n₈ on days 0, 5, 7, 12, and 14 (Injections ofF1aA-OVA-m₄-n₈ are labeled as “Gal-OVA” and shown as green arrows on thex-axis of FIG. 5 ). In order to determine the ability of F1aA-OVA-m₄-n₈to deplete serum OVA-specific antibodies, the mice were bled on day −1to establish an initial antibody titer and then subsequent bleeds werecarried out after each injection of F1aA-OVA-m₄-n₈ on days 2, 6, 9. 13,and 16. The antibody titer for each mouse was determined via ELISA. Theresults from this study show that F1aA-OVA-m₄-n₈ is able to depleteserum antibody levels in mice. For example, one day after the firstF1aA-OVA-m₄-n₈ injection (i.e. day 2), mice with positive OVA-antibodytiters experience a 5 to 100-fold decrease in serum antibody levels(FIG. 5 ). These results show that although over the course of the 19day experiment, antibody titers did increase for certain mice, the titerlevels never reached the initial antibody titer measured on Day −1 andsubsequent doses of F1aA-OVA-m₄-n₈ were effective in reducing thesetransient increases in antibody titers. These results demonstrate thatF1aA-OVA-m₄-n₈ has the specificity to bind serum OVA-specific antibodiesand the kinetics required to deplete OVA-specific serum antibodies.

13B. By following the procedure described in Example 13A andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formula 1 obtained,for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 15G, it isshown that the compounds from Examples 3A, 4A, 5B, 6C, 7B and 15G havethe specificity to bind serum OVA-specific antibodies and the kineticsrequired to deplete OVA-specific serum antibodies.

13C. By following the procedure described in Example 13A andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formula 1 obtained,for example, as described in Examples 1E, 1G, 2C, 10D, 15I, 15L, 16B,16D and 16F, and substituting OVA with the antigens corresponding to X(or X′ or X″), respectively, it is shown that the compounds fromExamples 1E, 1G, 2C, 15I, 15L, 16B, 16D and 16F have the specificity tobind serum antigen X-specific antibodies and the kinetics required todeplete antigen X-specific serum antibodies.

13D. By following the procedure described in Example 13A-C andsubstituting the corresponding glucosylated compounds for thegalactosylated compounds, it is confirmed that the glucolsylatedcompounds have the specificity to bind serum antigen X-specificantibodies and the kinetics required to deplete antigen X-specific serumantibodies.

Example 14 OT-1 Challenge-to-Tolerance Model

14A. Using an established OTI challenge-to-tolerance model (Liu, Iyoda,et al., 2002), the ability of F1aA-OVA-m₄-n₈ (mGal-OVA) andF1b-OVA-m₁-n₄-p₃₄ (pGal-OVA) to prevent subsequent immune responses tovaccine-mediated antigen challenge were demonstrated—even with achallenge involving a very strong bacterially-derived adjuvant (i.e.lipopolysaccharide). To tolerize, 233 nmol of either F1aA-OVA-m₄-n₈,F1b-OVA-m₁-n₄-p₃₄, or soluble OVA were intravenously administered in 100μl saline at 1 and 6 days following adoptive transfer of OTI CD8⁺(CD45.2⁺) T cells to CD45.1⁺ mice (n=5 mice per group). After 9additional days to allow potential deletion of the transferred T cells,the recipient mice were then challenged with OVA (10 μg) adjuvanted withlipopolysaccharide (LPS) (50 ng) by intradermal injection.Characterization of the draining lymph nodes 4 d after challenge alloweda determination as to whether or not deletion actually took place.

14B. Intravenous administration of F1aA-OVA-m₄-n₈ and F1b-OVA-m₁-n₄-p₃₄resulted in profound reductions in OTI CD8⁺ T cell populations in thedraining lymph nodes as compared to mice treated with unmodified OVAprior to antigen challenge with LPS, demonstrating deletional tolerance.For example, FIGS. 6A-6F show that the draining lymph nodes from micetreated with either F1aA-OVA-m₄-n₈ (mGal-OVA) and F1b-OVA-m₁-n₄-p₃₄(pGal-OVA) contained over 9-fold fewer OTI CD8⁺ T cells as compared toOVA-treated mice, and more than 43-fold fewer than the challenge controlmice that did not receive intravenous injections of antigen; responsesin spleen cells were similar. These results demonstrate thatF1aA-OVA-m₄-n₈ and F1b-OVA-m₁-n₄-p₃₄ mitigated an OVA-specific immuneresponse after adjuvented OVA challenge, thus establishing that thecompositions disclosed herein are suitable for induction of immunetolerance. As to characterization, FIG. 7 shows characterization ofF1aA-OVA-m₄-n₈₀ and F1b-OVA-m₁-n₄₄-p₃₄.

14C. By following the procedure described in Examples 14A and B, andsubstituting F1aA-OVA-m₄-n₈ and F1b-OVA-m₁-n₄-p₃₄ with the compounds ofFormula 1 obtained, for example, as described in Examples 3A, 4A, 5B,6C, 7B and 15G, it is shown that the compounds from Examples 3A, 4A, 5B,6C, 7B and 15G mitigate an OVA-specific immune response after adjuventedOVA challenge.

14D. By following the procedure described in Examples 14A and B, andsubstituting F1aA-OVA-m₄-n₈ and F1b-OVA-m₁-n₄-p₃₄ with the compounds ofFormula 1 obtained, for example, as described in Examples 1E, 1G, 2C,15I, 15L, 16B, 16D and 16F, and substituting OVA with the antigenscorresponding to X (or X′ or X″), respectively, it is shown that thecompounds from Examples 1E, 1G, 2C, 15I, 15L, 16B, 16D and 16F mitigatean antigen X-specific immune response after adjuvented antigen Xchallenge.

14E. By following the procedure described in Example 14A-D andsubstituting the corresponding glucosylated compounds for thegalactosylated compounds, it is confirmed that the glucolsylatedcompounds mitigate an antigen X-specific immune response afteradjuvanted antigen X challenge.

Example 15 F1m-OVA-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc

15A. Formula 1102 where R³ is NHAc and R⁴ is OH

N-Acetyl-D-galactosamine (Formula 1101 where R³ is NHAc and R⁴ is OH)(5g, 22.6 mmol) was added to a stirred solution of chloroethanol (200m₁) at room temperature. The solution was cooled to 4° C. andacetylchloride was added drop-wise to the solution. The solution wasbrought to room temperature and then heated to 70° C. After 4 hours, theunreacted choroethanol was removed under reduced pressure. 100 ml ofethanol was added to the crude product and the resulting solution wasstirred in the presence of carbon for 2 hours. The solution wasfiltered, and the solvent was removed under reduced pressure. Thecorresponding product of Formula 1102,N-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide,was used without further purification.

15B. Formula 1103 where R³ is NHAc and R⁴ is OH

TheN-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamideprepared in Example 15A (2g, 7.4 mmol) was added to a stirred solutionof DMF (100 m₁) and sodium azide (4g, 61.5 mmol). The solution washeaded at 90° C. for 12 hours and then filtered. The residual solventwas removed under reduced pressure and the crude product was purifiedvia flash chromatography (10% MeOH in dichloromethane) to give thecorresponding product of Formula 1103,N-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide.

15C. Formula 1104 where R³ is NHAc and R⁴ is OH

TheN-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamideprepared in Example 15B (2 g, 6.9 mmol) was added to a solution ofpalladium on carbon and ethanol (50 m₁). The solution was stirred underhydrogen gas (3 atm) for 4 hours. The resulting solution was filteredand the residual solvent was removed under reduced pressure to affordthe corresponding product of Formula 1104,N-(2-(2-aminoethoxy)-4,5-dihydroxy-6-(hydrownethyl)tetrahydro-2H-pyran-3-yl)acetamide,which was used without further purification.

15D. Formula 1105 where R³ is NHAc and R⁴ is OH

TheN-(2-(2-aminoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamideprepared in Example 15C (1.0 g, 3.78 mmol) was added to a solution ofmethacrylate anhydride (0.583 g, 3.78 mmol) in DMF (50 m₁).Triethylamine was then added to the solution and the reaction wasstirred for 2 hours at room temperature. After 2 hours, the excesssolvent was removed under reduced pressure, and the correspondingproduct of Formula 1105,N-(2-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)methacrylamide,was isolated via flash chromatography.

15E. Formula 1107 where p is 30, q is 4, R³ is NHAc, R⁴ is OH and R⁸ isCMP

An azide-modified uRAFT agent of Formula 1106 where q is 4 (28 mg) wasadded to a solution ofN-(2-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)methacrylamideprepared in Example 15D (579 mg, 1.74 mmol) and azobisisobutyronitrile(2.2 mg, 0.0116 mmol) in DMF. The reaction mixture was subjected to 4free-pump-thaw cycles, and then stirred at 70° C. After 12 hours, thepolymer product of Formula 1107, where p is 30 and q is 4 wasprecipitated from the reaction mixture via the addition of methanol. Thesolvent was decanted from the solid and the solid was collected andresidual solvent was removed via reduced pressure.

15F. Formula 1109 where X′ is OVA, m is 2 and n is 80

Ovalbumin (5 mg, 0.00012 mmol) was added to 100 μl of sodium phosphatebuffer (pH 8.0) and stirred. To this solution was added 5 mg of thecompound of Formula 1108 where n is 80. After 1 hour, the unreactedcompound of Formula 1108 was removed from the solution via centrifugalsize-exclusion chromatography. The resulting buffered solutioncontaining the corresponding product of Formula 1109 was used in thenext reaction without further purification.

15G. Formula 1m where X′ is OVA, m is 2, n is 80, p is 30, q is 4, R³ isNHAc and R⁸ is CMP

The solution prepared in Example 15F was added to 100 μl of sodiumphosphate buffer (pH 8.0) which contained 10 mg of the product ofFormula 1107 prepared in Example 15E. The reaction was allowed to stirfor 2 hours and then the excess Formula 1107 was removed via centrifugalsize exclusion chromatography to afford the corresponding isomericproduct of Formula 1m in solution, which was used in biological studieswithout further purification. The R³ substituent is shown in the name ofthe title compound as 2NHAc.

15H. Other Compounds of Formula 1109

By following the procedure described in Example 15F and substituting OVAwith the following:

-   -   Abciximab,    -   Adalimumab,    -   Agalsidase alfa,    -   Agalsidase beta,    -   Aldeslukin,    -   Alglucosidase alfa,    -   Factor VIII,    -   Factor IX,    -   L-asparaginase,    -   Laronidase,    -   Octreotide,    -   Phenylalanine ammonia-lyase,    -   Rasburicase,    -   Insulin (SEQ ID NO:1),    -   GAD-65 (SEQ ID NO:2),    -   IGRP (SEQ ID NO:3)    -   MBP (SEQ ID NO:4),    -   MOG (SEQ ID NO:5),    -   PLP (SEQ ID NO:6),    -   MBP13-32 (SEQ ID NO:7),    -   MBP83-99 (SEQ ID NO:8),    -   MBP111-129 (SEQ ID NO:9),    -   MBP146-170 (SEQ ID NO:10),    -   MOG1-20 (SEQ ID NO:11),    -   MOG35-55 (SEQ ID NO:12),    -   PLP139-154 (SEQ ID NO:13),    -   MART1 (SEQ ID NO:14),    -   Tyrosinase (SEQ ID NO:15),    -   PMEL (SEQ ID NO:16),    -   Aquaporin-4 (SEQ ID NO:17),    -   S-arrestin (SEQ ID NO:18),    -   IRBP (SEQ ID NO:19),    -   Conarachin (UNIPROT Q6PSU6),    -   Alpha-gliadin “33-mer” native (SEQ ID NO:20),    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21),    -   Alpha-gliadin (SEQ ID NO:22),    -   Omega-gliadin (SEQ ID NO:23),    -   Fel d 1A (UNIPROT P30438),    -   Cat albumin (UNIPROT P49064),    -   Can f 1 (UNIPROT 018873),    -   Dog albumin (UNIPROT P49822), and    -   RhCE (UNIPROT P18577),        there are obtained the following corresponding compounds of        Formula 1109 where n is 80:    -   X is Abciximab and m is 10,    -   X is Adalimumab and m is 11,    -   X is Agalsidase alfa and m is 14,    -   X is Agalsidase beta and m is 14,    -   X is Aldeslukin and m is 6,    -   X is Alglucosidase alfa and m is 13,    -   X is Factor VIII and m is 100,    -   X is Factor IX and m is 18,    -   X is L-asparaginase and m is 5,    -   X is Laronidase and m is 7,    -   X is Octreotide and m is 1,    -   X is Phenylalanine ammonia-lyase and m is 12,    -   X is Rasburicase and m is 12,    -   X is Insulin (SEQ ID NO:1) and m is 2,    -   X is GAD-65 (SEQ ID NO:2) and m is 8,    -   X is IGRP (SEQ ID NO:3) and m is 7,    -   X is MBP (SEQ ID NO:4) and m is 6,    -   X is MOG (SEQ ID NO:5) and m is 5,    -   X is PLP (SEQ ID NO:6) and m is 8,    -   X is MBP13-32 (SEQ ID NO:7) and m is 1,    -   X is MBP83-99 (SEQ ID NO:8) and m is 1,    -   X is MBP111-129 (SEQ ID NO:9) and m is 1,    -   X is MBP146-170 (SEQ ID NO:10) and m is 2,    -   X is MOG1-20 (SEQ ID NO:11) and m is 1,    -   X is MOG35-55 (SEQ ID NO:12) and m is 2,    -   X is PLP139-154 (SEQ ID NO:13) and m is 3,    -   X is MART1 (SEQ ID NO:14) and m is 4,    -   X is Tyrosinase (SEQ ID NO:15) and m is 8,    -   X is PMEL (SEQ ID NO:16) and m is 5,    -   X is Aquaporin-4 (SEQ ID NO:17) and m is 4,    -   X is S-arrestin (SEQ ID NO:18) and m is 12,    -   X is IRBP (SEQ ID NO:19) and m is 21,    -   X is Conarachin and m is 21,    -   X is Alpha-gliadin “33-mer” native (SEQ ID NO:20) and m is 1,    -   X is Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21) and m is        1,    -   X is Alpha-gliadin (SEQ ID NO:22) and m is 1,    -   X is Omega-gliadin (SEQ ID NO:23) and m is 1,    -   X is Fel d 1 and m is 4,    -   X is Cat albumin and m is 16,    -   X is Can f 1 and m is 6,    -   X is Dog albumin and m is 23, and    -   X is RhCE and m is 10.

15I. Other Compounds of Formula 1m

By following the procedure described in Example 15G and substituting thecompounds of Formula 1109, for example as obtained in Example 15H, thereare obtained the following corresponding compounds of Formula 1m:

-   -   F1m-Abciximab-m₁₀-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Adalimumab-m-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Agalsidase alfa-m₁₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Agalsidase beta-m₁₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Aldeslukin-m₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alglucosidase alfa-m₁₃-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Factor VIII-m₁₀₀-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Factor IX-m₁₈-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-L-asparaginase-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Laronidase-m₇-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Octreotide-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Phenylalanine ammonia-lyase-m₁₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Rasburicase-m₁₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Insulin-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-GAD-65-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-IGRP-m₇-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP-m₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MOG-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-PLP-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP13-32-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP83-99-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP111-129-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP146-170-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MOG1-20-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MOG35-55-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-PLP139-154-m₃-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MART1-m₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Tyrosinase-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-PMEL-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Aquaporin-4-m₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-S-arrestin-m₁₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-IRBP-m₂₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Conarachin-m₂₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alpha-gliadin “33-mer” native-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alpha-gliadin “33-mer” deamidated-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alpha-gliadin-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Omega-gliadin-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Fel d 1-m₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Cat albumin-m₁₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Can f 1-m₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Dog albumin-m₂₃-n₈₀-p₃₀-q₄-CMP-2NHAc, and    -   F1m-RhCE-m₁₀-n₈₀-p₃₀-q₄-CMP-2NHAc.

15J. Formula 1107 where p is 30, q is 8, R³ is OH, R⁴ is OH and R⁸ isCMP

By following the procedure described in Example 15A and substituting theN-acetyl-D-galactosamine with galactose, and following through to theprocedure described in Example 15E except using an azide-modified uRAFTagent of Formula 1106 where q is 8, there is obtained the compound ofFormula 1107 where p is 30, q is 8, R³ is OH, R⁴ is OH and R⁸ is CMP.

15K. Formula 1109 where n is 62 and where X′ and m are as in Example 19H

By following the procedure described in Example 15F, substituting theOVA with the compounds as described in Example 15H and employing thecompound of Formula 1108 where n is 62, there are obtained thecorresponding compounds of Formula 1109 where n is 62.

15L. Other Compounds of Formula 1m

By following the procedure described in Example 15G and substituting thecompound of Formula 1107 with the compounds obtained in Example 15J, andsubstituting the compound of Formula 1109 with the compounds obtained inExample 15K, there are obtained the following corresponding compounds ofFormula 1m:

-   -   F1m-Abciximab-m₁₀-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Adalimumab-m₁₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Agalsidase alfa-m₁₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Agalsidase beta-m₁₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Aldeslukin-m₆-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alglucosidase alfa-m₁₃-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Factor VIII-m₁₀₀-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Factor IX-m₁₈-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-L-asparaginase-m₅-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Laronidase-m₇-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Octreotide-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Phenylalanine ammonia-lyase-m₁₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Rasburicase-m₁₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Insulin-m₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-GAD-65-m₅-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-IGRP-m₇-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP-m₆-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MOG-m₅-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-PLP-m₅-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP13-32-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP83-99-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP111-129-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP146-170-m₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MOG1-20-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MOG35-55-m₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-PLP139-154-m₃-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MART1-m₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Tyrosinase-m₅-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-PMEL-m₅-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Aquaporin-4-m₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-S-arrestin-m₁₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-IRBP-m₂₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Conarachin-m₂₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alpha-gliadin “33-mer” native-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alpha-gliadin “33-mer” deamidated-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alpha-gliadin-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Omega-gliadin-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Fel d 1-m₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Cat albumin-m₁₆-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Can f 1-m₆-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Dog albumin-m₂₃-n₆₂-p₃₀-q₈-CMP-2OH, and    -   F1m-RhCE-m₁₀-n₆₂-p₃₀-q₈-CMP-2OH.

15M. Other Compounds of Formula 1m

By following the procedure described in Examples 15A-L and substitutingthe galactosamine or galactose with glucosamine or glucose,respectively, there are obtained the corresponding glucosylatedcompounds of Formula 1m.

Example 16 F1n-insulin-m₂-n₁-p₃₀-q₄-CMP-2NHAc

16A. Formula 1202 where X′ is Insulin, m is 2 and n is 1

Recombinant human insulin (5 mg) was added to 100 μl of DMF containing10 μl of triethylamine and stirred until the insulin became soluble. Tothis solution was added 10 mg (0.0161 mmol) of a linker precursor ofFormula 1201 where n is 1 and the reaction was allowed to stir. After 1hour, 1.3 ml of tert-butyl methyl ether was added to isolate thecorresponding product of Formula 1202, which was recovered as theprecipitate. Residual DMF and tert-butyl methyl ether were removed underreduced pressure. Characterization via liquid chromatography, massspectroscopy and polyacrylamide gel electrophoresis confirmed theidentity of the product. The modified insulin product of Formula 1202was used without further purification.

16B. Formula 1n where X′ is Insulin, m is 2, n is 1, p is 30, q is 4 andR⁸ is CMP

The product of Formula 1202 obtained in Example 16A was resuspended in100 μl of DMF. The polymer product of Formula 1107 obtained in Example15E (10 mg) was added and the reaction was allowed to stir for 1 hour.After 1 hour, the reaction products were precipitated via the additionof dichloromethane (1.3 ml). The product was filtered and the residualsolvent was removed under reduced pressure. The crude product was thenresuspended in 500 μl of PBS, and the low molecular weight componentswere removed via centrifugal size exclusion chromatography to afford thecorresponding isomeric product of Formula 1n. Characterization vialiquid chromatography, mass spectroscopy and polyacrylamide gelelectrophoresis confirmed the identity of the product. The modifiedinsulin product of Formula 1202 was used without further purification.

16C. Other Compounds of Formula 1202

By following the procedure described in Example 16A and substitutinginsulin with the following:

-   -   Abciximab,    -   Adalimumab,    -   Agalsidase alfa,    -   Agalsidase beta,    -   Aldeslukin,    -   Alglucosidase alfa,    -   Factor VIII,    -   Factor IX,    -   L-asparaginase,    -   Laronidase,    -   Octreotide,    -   Phenylalanine ammonia-lyase,    -   Rasburicase,    -   GAD-65 (SEQ ID NO:2),    -   IGRP (SEQ ID NO:3)    -   MBP (SEQ ID NO:4),    -   MOG (SEQ ID NO:5),    -   PLP (SEQ ID NO:6),    -   MBP13-32 (SEQ ID NO:7),    -   MBP83-99 (SEQ ID NO:8),    -   MBP111-129 (SEQ ID NO:9),    -   MBP146-170 (SEQ ID NO:10),    -   MOG1-20 (SEQ ID NO:11),    -   MOG35-55 (SEQ ID NO:12),    -   PLP139-154 (SEQ ID NO:13),    -   MART1 (SEQ ID NO:14),    -   Tyrosinase (SEQ ID NO:15),    -   PMEL (SEQ ID NO:16),    -   Aquaporin-4 (SEQ ID NO:17),    -   S-arrestin (SEQ ID NO:18),    -   IRBP (SEQ ID NO:19),    -   Conarachin (UNIPROT Q6PSU6),    -   Alpha-gliadin “33-mer” native (SEQ ID NO:20),    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21),    -   Alpha-gliadin (SEQ ID NO:22),    -   Omega-gliadin (SEQ ID NO:23),    -   Fel d 1A (UNIPROT P30438),    -   Cat albumin (UNIPROT P49064),    -   Can f 1 (UNIPROT 018873),    -   Dog albumin (UNIPROT P49822), and    -   RhCE (UNIPROT P18577),        there are obtained the following corresponding compounds of        Formula 1202 where n is 1:    -   X is Abciximab and m is 10,    -   X is Adalimumab and m is 11,    -   X is Agalsidase alfa and m is 14,    -   X is Agalsidase beta and m is 14,    -   X is Aldeslukin and m is 6,    -   X is Alglucosidase alfa and m is 13,    -   X is Factor VIII and m is 100,    -   X is Factor IX and m is 18,    -   X is L-asparaginase and m is 5,    -   X is Laronidase and m is 7,    -   X is Octreotide and m is 1,    -   X is Phenylalanine ammonia-lyase and m is 12,    -   X is Rasburicase and m is 12,    -   X is GAD-65 (SEQ ID NO:2) and m is 8,    -   X is IGRP (SEQ ID NO:3) and m is 7,    -   X is MBP (SEQ ID NO:4) and m is 6,    -   X is MOG (SEQ ID NO:5) and m is 5,    -   X is PLP (SEQ ID NO:6) and m is 8,    -   X is MBP13-32 (SEQ ID NO:7) and m is 1,    -   X is MBP83-99 (SEQ ID NO:8) and m is 1,    -   X is MBP111-129 (SEQ ID NO:9) and m is 1,    -   X is MBP146-170 (SEQ ID NO:10) and m is 2,    -   X is MOG1-20 (SEQ ID NO:11) and m is 1,    -   X is MOG35-55 (SEQ ID NO:12) and m is 2,    -   X is PLP139-154 (SEQ ID NO:13) and m is 3,    -   X is MART1 (SEQ ID NO:14) and m is 4,    -   X is Tyrosinase (SEQ ID NO:15) and m is 8,    -   X is PMEL (SEQ ID NO:20) and m is 5,    -   X is Aquaporin-4 (SEQ ID NO:21) and m is 4,    -   X is S-arrestin (SEQ ID NO:22) and m is 12,    -   X is IRBP (SEQ ID NO:19) and m is 21,    -   X is Conarachin and m is 21,    -   X is Alpha-gliadin “33-mer” native (SEQ ID NO:20) and m is 1,    -   X is Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21) and m is        1,    -   X is Alpha-gliadin (SEQ ID NO:22) and m is 1,    -   X is Omega-gliadin (SEQ ID NO:27) and m is 1,    -   X is Fel d 1 and m is 4,    -   X is Cat albumin and m is 16,    -   X is Can f 1 and m is 6,    -   X is Dog albumin and m is 23, and    -   X is RhCE and m is 10.

16D. Other Compounds of Formula 1n

By following the procedure described in Example 16B and substituting thecompounds of Formula 1202, for example as obtained in Example 16C, thereare obtained the following corresponding compounds of Formula 1m:

-   -   F1n-Abciximab-m₁₀-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Adalimumab-m₁₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Agalsidase alfa-m₁₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Agalsidase beta-m₁₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Aldeslukin-m₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alglucosidase alfa-m₁₃-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Factor VIII-m₁₀₀-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Factor IX-m₁₈-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-L-asparaginase-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Laronidase-m₇-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Octreotide-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Phenylalanine ammonia-lyase-m₁₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Rasburicase-m₁₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-GAD-65-m₅-n i-p₃₀-q₄-CMP-2NHAc,    -   F1n-IGRP-m₇-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP-m₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MOG-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-PLP-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP13-32-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP83-99-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP111-129-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP146-170-m₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MOG1-20-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MOG35-55-m₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-PLP139-154-m₃-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MART1-m₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Tyrosinase-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-PMEL-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Aquaporin-4-m₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-S-arrestin-m₁₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-IRBP-m₂₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Conarachin-m₂₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alpha-gliadin “33-mer” native-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alpha-gliadin “33-mer” deamidated-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alpha-gliadin-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Omega-gliadin-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Fel d 1-m₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Cat albumin-m₁₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Can f 1-m₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Dog albumin-m₂₃-n₁-p₃₀-q₄-CMP-2NHAc, and    -   F1n-RhCE-m₁₀-n₁-p₃₀-q₄-CMP-2NHAc.

16E. Formula 1202 where n is 33 and where X′ and m are as in Example 20C

By following the procedure described in Example 16F, substituting theinsulin with the compounds as described in Example 16C and employing thecompound of Formula 1201 where n is 33, there are obtained thecorresponding compounds of Formula 1202 where n is 33.

16F. Other Compounds of Formula 1n

By following the procedure described in Example 16B and substituting thecompound of Formula 1107 with the compounds obtained in Example 15J, andsubstituting the compound of Formula 1202 with the compounds obtained inExample 16E, there are obtained the following corresponding compounds ofFormula 1n:

-   -   F1n-Abciximab-m₁₀-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Adalimumab-m₁₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Agalsidase alfa-m₁₄-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Agalsidase beta-m₁₄-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Aldeslukin-m₆-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Alglucosidase alfa-m₁₃-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Factor VIII-m₁₀₀-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Factor IX-m₁₈-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-L-asparaginase-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Laronidase-m₇-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Octreotide-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Phenylalanine ammonia-lyase-m₁₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Rasburicase-m₁₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-GAD-65-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-IGRP-m₇-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP-m₆-n₃₃-p₃₀-co-CMP-2OH,    -   F1n-MOG-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-PLP-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP13-32-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP83-99-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP111-129-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP146-170-m₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MOG1-20-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MOG35-55-m₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-PLP139-154-m₃-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MART1-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Tyrosinase-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-PMEL-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Aquaporin-4-m₄-n₃₃-p₃₀-co-CMP-2OH,    -   F1n-S-arrestin-m₁₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-IRBP-m₂₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Conarachin-m₂₁-n₃₃-p₃₀-co-CMP-2OH,    -   F1n-Alpha-gliadin “33-mer” native-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Alpha-gliadin “33-mer” deamidated-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Alpha-gliadin-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Omega-gliadin-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Fel d 1-m₄-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Cat albumin-m₁₆-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Can f 1-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Dog albumin-m₂₃-n₃₃-p₃₀-q₈-CMP-2OH, and    -   F1n-RhCE-m₁₀-n₃₃-p₃₀-q₈-CMP-2OH.

16G. Other Compounds of Formula 1n

By following the procedure described in Examples 16A-F and substitutingthe galactosylating moieties with glucosylating moieties, there areobtained the corresponding glucosylated compounds of Formula 1n.

Example 17 Formula 1507 where p is 90, t is 1, R³ is NHAc and R⁴ is OH

17A. Formula 1502 where t is 1, R³ is NHAc and R⁴ is OH

N-Acetyl-D-glucosamine (Formula 1101 where R³ is NHAc and R⁴ is OH) (5.0g, 22.6 mmol) was added to a stirred solution of2-(2-chloroethoxy)ethan-1-ol (50 m₁) at room temperature. The solutionwas cooled to 4° C. and acetylchloride was added drop-wise to thesolution. The solution was brought to room temperature and then heatedto 70° C. After 4 hours, the reaction mixture was added to 200 ml ofethyl acetate. The precipitate that formed was collected, added to 100ml of ethanol and stirred in the presence of carbon for 2 hours. Thesolution was filtered, and the solvent was removed under reducedpressure. The corresponding product of Formula 1502,N-acetyl-D-glucosamine-2-(chloroethoxy)ethanol, was used without furtherpurification.

17B. Formula 1503 where t is 1, R³ is NHAc and R⁴ is OH

N-Acetyl-D-glucosamine-2-(chloroethoxy)ethanol (2.0 g, 6.11 mmol) wasadded to a stirred solution of DMF (100 m₁) and sodium azide (4.0 g,61.5 mmol). The solution was headed at 90° C. for 12 hours and thenfiltered. The residual solvent was removed under reduced pressure andthe crude product was purified via flash chromatography (10% MeOH indichloromethane) to give the corresponding product of Formula 1503,N-acetyl-D-glucosamine-2-(azideoethoxy)ethanol.

17C. Formula 1504 where t is 1, R³ is NHAc and R⁴ is OH

N-Acetyl-D-glucosamine-2-(azideoethoxy)ethanol (2.0 g, 5.9 mmol) wasadded to a solution of palladium on carbon and ethanol (50 m₁). Thesolution was stirred under hydrogen gas (3 atm) for 4 hours. Theresulting solution was filtered and the residual solvent was removedunder reduced pressure to afford the corresponding product of Formula1504, N-acetyl-D-glucosamine-2-(amineoethoxy)ethanol.

17D. Formula 1505 where t is 1, R³ is NHAc and R⁴ is OH

N-Acetyl-D-glucosamine-2-(amineoethoxy)ethanol (1.0 g, 3.25 mmol) wasadded to a solution of methacrylate anhydride (0.583 g, 3.78 mmol) inDMF (50 m₁). Triethylamine was then added to the solution and thereaction was stirred for 2 hours at room temperature. After 2 hours, theexcess solvent was removed under reduced pressure, and the correspondingproduct of Formula 1505,((2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-2-(2-(2-methacrylamidoethoxy)ethoxy)tetrahydro-2H-pyran-3-yl)carbamicacid, was isolated via flash chromatography.

17E. Formula 1507 where p is 90, q is 4, t is 1, R³ is NHAc, R⁴ is OH,R⁸ is CMP, and R¹⁰ is 2-hydroxypropyl

A 25 ml Schlenk flask was charged with a compound of Formula 1505, theproduct of Example 17D (272 mg, 0.72 mmol),N-(2-hydroxypropyl)methacrylamide (“HPMA”, used as received from themanufacturer) (211 mg, 1.47 mmol), an azide-modified uRAFT agent ofFormula 1106 where q is 4 and R⁸ is CMP (10.41 mg, 0.0217 mmol),azobis(isobutyronitril) (0.98 mg, 0.005 mmol), and 1.2 mldimethylformamide. The reaction mixture was subjected to fourfreeze-pump-thaw degassing cycles and then stirred at 70° C. for 20hours. The corresponding random polymeric product of Formula 1507 wasrecovered by precipitating the reaction mixture in acetone. Excessacetone was removed at reduced pressure to provide the random polymericproduct, which was used without further purification.

17F. Formula 1507 where p is 90, q is 4, t is 1, R³ is NHAc, R⁴ is OH,R⁸ is CMP, and R¹⁰ is 2-hydroxypropyl, using N-acetyl-D-galactosamine

By following the procedures of Examples 17A through 17E and substitutingN-acetyl-D-galactosamine for N-acetyl-D-glucosamine in the procedure ofExample 17A, there was obtained the corresponding galactosyl compound ofFormula 1507.

17G. Compounds of Formula 1507 where t is Other than 1

By following the procedures of Examples 17A through 17E and substituting2-(2-chloroethoxy)ethan-1-ol with:

-   2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol will afford the corresponding    compound of Formula 1507 where t is 2,-   2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethan-1-ol will afford the    corresponding compound of Formula 1507 where t is 3,-   2-(2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethoxy)ethan-1-ol will    afford the corresponding compound of Formula 1507 where t is 4,-   2-(2-(2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethan-1-ol    will afford the corresponding compound of Formula 1507 where t is 5,    and-   2-(2-(2-(2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethan-1-ol    will afford the corresponding compound of Formula 1507 where t is 6.

17H. Compounds of Formula 1507 Having a Plurality of W¹ Groups where tVaries

By following the procedure of Example 17E and substituting the compoundof Formula 1505 where t is 1 with 0.36 mmol each of Formula 1505 where tis 2 and 4, (prepared, for example, as described in Example 17F byfollowing the procedures of Examples 17A through 17D) there is obtainedthe corresponding random copolymer of Formula 1507 having about 15 W¹groups where t is 2, 15 W¹ groups where t is 4 and 60 W² groups.

17I. Compounds of Formula 1507 Having a Mixture of Glucosyl andGalactosyl Moieties

By following the procedure of Example 17E and substituting the compoundof Formula 1505 with 0.36 mmol each of glucosyl and galactosyl Formula1505 (prepared, for example, as described in Example 17D and in Example17F by following the procedures of Examples 17A through 17D) there isobtained the corresponding random copolymer of Formula 1507 having about15 glucosyl W¹ groups, 15 galactosyl W¹ groups and 60 W² groups.

Example 17.1 Formula 1507 where t is 1, R³ is NHAc and R⁴ is OH

17.1A. Formula 1502 where t is 1, R³ is NHAc and R⁴ is OH:2-(2-(2-chloroethoxy)ethoxy)-α-NAc-Galactosamine (1502.1A).

Acetyl chloride (4.35 mL, 61.05 mmol) was added dropwise to the ice-coldsolution of NHAc protected D-Galactosamine (10.0 g) in2-(2′-Chloroethoxy)ethanol (40 mL). The mixture was stirred for 15minutes at 4° C. and then was transferred to the oil bath at 70° C. Thereaction was left mixing under cooling condenser for 4 hours. After thattime, a dark brown solution was cooled down and poured into 400 mLsolution of ethyl acetate and dichloromethane (3:1, v/v) in order to getrid of an excess of unreacted chloroethanol. The mixture was placed in afreezer for 30 minutes and then decanted from dark brown, stickyprecipitate. The precipitate was dissolved in anhydrous ethanol andactivated charcoal was added. The suspension was mixed for 1.5 hours andthen filtered off through Celite and washed with ethanol. In the laststep, ethanol was evaporated in vacuum to provide 12.8 g of product(1502.1A) (95.24% yield).

17.1B. Formula 1503 where t is 1, R³ is NHAc and R⁴ is OH;2-(2-(2-Azidoethoxy)ethoxy)-α-NAc-Galactosamine (1503.16).

A compound (1502.1A) (5.0 g) was dissolved in 20 mL ofN,N-dimethylformamide. To that solution, sodium azide (26628-22-8) wasadded (5.0 g). The suspension was placed in an oil bath and stirred overnight at 80° C. After the night, the reaction mixture was filtered offthrough Celite. The solvent was then evaporated under high pressure toprovide an oily, brown substance. Final product was purified via flashchromatography (82.2% yield).

17.1C. Formula 1504 where t is 1, R³ is NHAc and R⁴ is OH;2-(2-(2-aminoethoxy)ethoxy)-α-NAc-Galactosamine (1504.1C).

A suspension of (1503.1B) (5.5 g) and 10% palladium on carbon (ca. 500mg) in 20 mL of ethanol was hydrogenated in a Shlenk flask with aninitial pressure of 2 bars of hydrogen gas. The reduction process wascontrolled by TLC. After 3 hours reaction was completed and thesuspension was filtered through Celite (78% yield).

17.1D. Formula 1505 where t is 1, R³ is NHAc and R⁴ is OH;α-NAc-Glactosamine-amine-methacrylate (1505.1 D)

A compound (1504.1C) (4.5 g) was dissolved in 10 mL ofN,N-dimethylformamide. To that solution, triethylamine (3 mL) was addedand the mixture was cooled down to 4° C. Subsequently, pentafluorophenylmethacrylate (13642-97-2) (4.38 mL) was added drop-wise with constantstirring. After 30 minutes, ice-bath was removed and the reaction wasallowed to stir at room temperature for the next 4 hours. Next, thesolvent was evaporated and the residual was adsorbed on silica gel. Thepurification of crude material using flash chromatography(dichloromethane:methanol 95:5, v/v) provided 3.8g of NAc-Galactosaminemonomer (α-NAc-Glactosamine-amine-methacrylate (1505.1D)) (64.73%yield).

Tetraethylene glycol mono p-toluenesulfonate (1651a).

Tetraethylene glycol (1650a) (112-60-7) (2.5 q) and pyridine (1.0 q)were added to 50 mL of dichloromethane and stirred for 20 minutes at 0°C. To that solution, p-toluenesulfonyl chloride (98-59-9)(2.37) in 15 mLof dichloromethane was added slowly. The reaction mixture was thenstirred for 2h at 0° C. followed by 4h at room temperature. After thattime, the solvent was evaporated and crude product was purified viaflash chromatography (ethyl acetate:hexane 6:4, v/v) to afford 1651a(44% yield).

S-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]ester (1652a)

To a suspension of potassium thioacetate (10387-40-3) (10.1 g, 88 mmol)in 650 mL of DMF was added a solution of (1651a) (15.4 g) in 100 mL ofDMF. The mixture was stirred at room temperature for 1 h and then at 90C for 4 h. After filtration, the solvent was evaporated under reducedpressure. The residue was dissolved in ethyl acetate (150 mL) and washedwith water (2×50 mL) and brine (2×50 mL). The aqueous wash solutionswere reextracted with ethyl acetate (2×50 mL), and the combined organiclayers were dried over magnesium sulfate and evaporated under reducedpressure to give a yellow oil product 1652a (45% yield)

2-(2-(2-(2-(pyridin-2-yldisulfanyl)ethoxy)ethoxy)ethoxy)ethan-1-ol(1653a)

Sodium methoxide (1.40 ml of 0.5M in methanol) was added dropwise to astirred solution of (1652a) (70.9 mg) and 2,2-dithiodipyridine(2127-03-9) (77.4 mg, 0.351 mmol) in anhydrous methanol (3 mL) under anargon atmosphere. After 2 h the reaction was concentrated with silica toa powder, and the crude product was purified by flash chromatographyover silica (1:1 hexanes:EtOAc) to afford 1653a as a clear, pale yellowliquid (26.3 mg, 44% yield).

uRAFT Aunt (1601a)

Compound 1653a (1g) was added dropwise to a stirred solution of4-Cyano-4-(thiobenzoylthio)pentanoic acid (1.1g) (201611-92-9),N,N′-Dicyclohexylcarbodiimide (538-75-0) (0.5 g) and4-Dimethylaminopyridine (DMAP) (1122-58-3) (0.1 g) in DCM (15m₁). Thereaction was stirred at 0 C for 2 h then allowed to warm to roomtemperature. After 3 h, the reaction was filtered through celite and thesolvent was removed via reduced pressure. The final product (1601a) wasrecovered from flash chromatography (67% yield).

pGal (17.1E)

The following conditions were performed using theα-NAc-Glactosamine-amine-methacrylate (e.g., 1505.1D) monomer to afford17.1E. In some embodiments, the α-NAc-Glucosamine-amine-methacrylatemonomer (e.g., 1505.2D) can be used instead to afford aglucosamine-based polymer. In some embodiments, a is an integer betweenabout 0 to about 150, about 1 to about 100, about 1 to about 50, about 1to about 10, or about 1 to about 5. In some embodiments, b is an integerbetween about 0 to about 150, about 1 to about 100, about 1 to about 50,about 1 to about 10, or about 1 to about 5.

Compound 1601a, 1505.1D, Azobisisobutyronitrile (78-67-1), andN-(2-hydroxypropyl)methacrylamide (21442-01-3) were added to DMF (1 ml).The reaction mixture was subjected to 4 freeze-pump-thaw degassingcycles before being stirred for 20 h at 70 C. The polymeric product wasrecovered via precipitation from acetone. The excess solvent was removedunder reduced pressure (55% yield).

Example 17.2 Formula 1507 where t is 1, R³ is NHAc and R⁴ is OH

17.2A. Formula 1502 where t is 1, R³ is NHAc and R⁴ is OH;2-(2-(2-chloroethoxy)ethoxy)-α-NAc-glucosamine (1502.2A).

Acetyl chloride (75-36-5) (4.35 mL, 61.05 mmol) was added dropwise tothe ice-cold solution of D-Glucosamine (7512-17-6) (10.0 g) in2-(2′-Chloroethoxy)ethanol (628-89-7) (40 mL). The mixture was stirredfor 15 minutes at 4° C. and then was transferred to the oil bath at 70°C. The reaction was left mixing under cooling condenser for 4 hours.After that time, a dark brown solution was cooled down and poured into400 mL solution of ethyl acetate and dichloromethane (3:1, v/v) in orderto get rid of an excess of unreacted chloroethanol. The mixture wasplaced in a freezer for 30 minutes and then decanted from dark brown,sticky precipitate. The precipitate was dissolved in anhydrous ethanoland activated charcoal was added. The suspension was mixed for 1.5 hoursand then filtered off through Celite and washed with ethanol. In thelast step, ethanol was evaporated in vacuum to afford 1502.2A (76%yield).

17.2B. Formula 1503 where t is 1, R³ is NHAc and R⁴ is OH;2-(2-(2-Azidoethoxy)ethoxy)-α-NAc-Glucosamine (1503.2B).

A compound (1502.2A) (5.0 g) was dissolved in 20 mL ofN,N-dimethylformamide. To that solution, sodium azide (26628-22-8) wasadded (5.0 g). The suspension was placed in an oil bath and stirred overnight at 80° C. After the night, the reaction mixture was filtered offthrough Celite. The solvent was then evaporated under high pressure toprovide an oily, brown substance. The final product 1503.26 was purifiedvia flash chromatography (75.4% yield).

17.2C. Formula 1504 where t is 1, R³ is NHAc and R⁴ is OH;2-(2-(2-aminoethoxy)ethoxy)-α-NAc-Glucosamine (1504.2C).

A suspension of (1503.2B) (5.5 g) and 10% palladium on carbon (ca. 500mg) in 20 mL of ethanol was hydrogenated in a Shlenk flask with aninitial pressure of 2 bars of hydrogen gas. The reduction process wascontrolled by TLC. After 3 hours reaction was completed and thesuspension was filtered through Celite to afford 1504.2C (65% yield).

17.2D. Formula 1505 where t is 1, R³ is NHAc and R⁴ is OH;α-NAc-Glucosamine-amine-methacrylate (1505.2D).

Compound 1504.2C (4.5 g) was dissolved in 10 mL ofN,N-dimethylformamide. To that solution, triethylamine (3 mL) was addedand the mixture was cooled down to 4° C. Subsequently, pentafluorophenylmethacrylate (13642-97-2) (4.38 mL) was added drop-wise with constantstirring. After 30 minutes, ice-bath was removed and the reaction wasallowed to stir at room temperature for the next 4 hours. Next, thesolvent was evaporated and the residual was adsorbed on silica gel. Thepurification of crude material using flash chromatography(dichloromethane:methanol 95:5, v/v) provided 3.8g of NAc-Glucosaminemonomer 1505.2D (74% yield).

Example 18 Formula 1m′ where X′ is OVA, m is 1-3, n is 79, p is 90 (30W¹+60 W²), q is 4, t is 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is2-hydroxypropyl

18A. Formula 1109 where X′ is OVA, m is 1-3 and n is 79

A solution of Formula 101′ where X′ is OVA (10 mg of endotoxin-freeovalbumin) in pH 7.6 PBS was added to Formula 1108 where n Is 79 (10 mg)in an endotoxin-free tube. The reaction mixture was allowed to stir atroom temperature. After 1 hour, any unconjugated Formula 1108 wasremoved via centrifugal size exclusion chromatography to afford thecorresponding product of Formula 1109, which was used without furtherpurification.

18B. Formula 1m′ where X′ is OVA, m is 1-3, n is 79, p is 90, q is 4, tis 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is 2-hydroxypropyl

The Formula 1109 solution obtained in Example 18A was then added toFormula 1507 as obtained in Example 17E (20 mg) in an endotoxin-freetube and stirred at room temperature to afford the corresponding productof Formula 1m′(“F1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀”),which was purified from the reaction mixture via fast protein liquidchromatography (FPLC) using a Superdex 200 prep grade column and usedwithout further purification.

18C. Formula 1m′ where X′ is OVA, m is 1-3, n is 79, p is 90, q is 4, tis 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is 2-hydroxypropyl, usingN-acetyl-D-galactosamine

By following the procedure of Example 18B and substituting thegalactosyl compound of Formula 1507 as obtained in Example 17F there wasobtained the corresponding galactosyl compound of Formula 1m′(“F1m′-OVA-m₁₋₃-n₇₉-p₉₀-q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀”).

18D. Other Compounds of Formula 1m′ where X′ is OVA, m is 1-3, n is 79,p is 90, q is 4, t is 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is2-hydroxypropyl

By following the procedures described in Example 18A, 18B and 18C andsubstituting OVA with the following:

Abciximab,

-   -   Adalimumab,    -   Agalsidase alfa,    -   Agalsidase beta,    -   Aldeslukin,    -   Alglucosidase alfa,    -   Factor VIII,    -   Factor IX,    -   L-asparaginase,    -   Laronidase,    -   Octreotide,    -   Phenylalanine ammonia-lyase,    -   Rasburicase,    -   GAD-65 (SEQ ID NO:2),    -   IGRP (SEQ ID NO:3)    -   MBP (SEQ ID NO:4),    -   MOG (SEQ ID NO:5),    -   PLP (SEQ ID NO:6),    -   MBP13-32 (SEQ ID NO:7),    -   MBP83-99 (SEQ ID NO:8),    -   MBP111-129 (SEQ ID NO:9),    -   MBP146-170 (SEQ ID NO:10),    -   MOG1-20 (SEQ ID NO:11),    -   MOG35-55 (SEQ ID NO:12),    -   PLP139-154 (SEQ ID NO:13),    -   MART1 (SEQ ID NO:14),    -   Tyrosinase (SEQ ID NO:15),    -   PMEL (SEQ ID NO:16),    -   Aquaporin-4 (SEQ ID NO:17),    -   S-arrestin (SEQ ID NO:18),    -   IRBP (SEQ ID NO:19),    -   Conarachin (UNIPROT Q6PSU6),    -   Alpha-gliadin “33-mer” native (SEQ ID NO:20),    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21),    -   Alpha-gliadin (SEQ ID NO:22),    -   Omega-gliadin (SEQ ID NO:23),    -   Fel d 1A (UNIPROT P30438),    -   Cat albumin (UNIPROT P49064),    -   Can f 1 (UNIPROT 018873),    -   Dog albumin (UNIPROT P49822), and    -   RhCE (UNIPROT P18577),        there are obtained the following corresponding glucosyl and        galactosyl compounds of Formula 1m′:    -   X′ is Abciximab and m is 10,    -   X′ is Adalimumab and m is 11,    -   X′ is Agalsidase alfa and m is 14,    -   X′ is Agalsidase beta and m is 14,    -   X′ is Aldeslukin and m is 6,    -   X′ is Alglucosidase alfa and m is 13,    -   X′ is Factor VIII and m is 100,    -   X′ is Factor IX and m is 18,    -   X′ is L-asparaginase and m is 5,    -   X′ is Laronidase and m is 7,    -   X′ is Octreotide and m is 1,    -   X′ is Phenylalanine ammonia-lyase and m is 12,    -   X′ is Rasburicase and m is 12,    -   X′ is GAD-65 (SEQ ID NO:2) and m is 8,    -   X′ is IGRP (SEQ ID NO:3) and m is 7,    -   X′ is MBP (SEQ ID NO:4) and m is 6,    -   X′ is MOG (SEQ ID NO:5) and m is 5,    -   X′ is PLP (SEQ ID NO:6) and m is 8,    -   X′ is MBP13-32 (SEQ ID NO:7) and m is 1,    -   X′ is MBP83-99 (SEQ ID NO:8) and m is 1,    -   X′ is MBP111-129 (SEQ ID NO:9) and m is 1,    -   X′ is MBP146-170 (SEQ ID NO:10) and m is 2,    -   X′ is MOG1-20 (SEQ ID NO:11) and m is 1,    -   X′ is MOG35-55 (SEQ ID NO:12) and m is 2,    -   X′ is PLP139-154 (SEQ ID NO:13) and m is 3,    -   X′ is MART1 (SEQ ID NO:14) and m is 4,    -   X′ is Tyrosinase (SEQ ID NO:15) and m is 8,    -   X′ is PMEL (SEQ ID NO:16) and m is 5,    -   X′ is Aquaporin-4 (SEQ ID NO:17) and m is 4,    -   X′ is S-arrestin (SEQ ID NO:18) and m is 12,    -   X′ is IRBP (SEQ ID NO:19) and m is 21,    -   X′ is Conarachin and m is 21,    -   X′ is Alpha-gliadin “33-mer” native (SEQ ID NO:20) and m is 1,    -   X′ is Alpha-gliadin “33-mer” deamidated (SEQ ID NO:21) and m is        1,    -   X′ is Alpha-gliadin (SEQ ID NO:22) and m is 1,    -   X′ is Omega-gliadin (SEQ ID NO:23) and m is 1,    -   X′ is Fel d 1 and m is 4,    -   X′ is Cat albumin and m is 16,    -   X′ is Can f 1 and m is 6,    -   X′ is Dog albumin and m is 23, and    -   X′ is RhCE and m is 10.

18E. Compounds of Formulae 1h′, 1i′, 1j′, 1k′, 1L′, and 1n′

By following the procedures described in Example 18B, 18C and 18D andsubstituting Formula 1109 with the following:

-   -   Formula 802 will afford the corresponding random copolymers of        Formula 1h′,    -   Formula 902 will afford the corresponding random copolymers of        Formula 1i′,    -   Formula 902 made with a compound of Formula 103′ will afford the        corresponding random copolymers of Formula 1j′,    -   Formula 1002 will afford the corresponding random copolymers of        Formula 1k′,    -   Formula 1002 made with a compound of Formula 103′ will afford        the corresponding random copolymers of Formula 1L′, and    -   Formula 1202 will afford the corresponding random copolymers of        Formula 1n′.

18F. Other Compounds of Formulae 1h′, 1i′, 1j′, 1k′, 1L′, 1m′ and 1n′

By following the procedures described in Example 18B, 18C, 18D and 18E,and substituting Formula 1507 with the compounds prepared as describedin Examples 17G, 17H and 17I, there are obtained the correspondingcompounds of Formulae 1h′, 1i′, 1j′, 1k′, 1L′, 1m′ and 1n′ where t isother than 1, having a plurality oft groups, and having a mixture ofglucosyl and galactosyl moieties.

Example 19 Formula 1c′ where X″ is Insulin-B, m is 1, n is 4, p is 90(30 W¹+60 W²), t is 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is2-hydroxypropyl

19A. Formula 1602 where n is 4, p is 90, t is 1, R³ is NHAc, R⁴ is OH,R⁸ is CMP, and R¹⁰ is 2-hydroxypropyl

A 25 ml Schlenk flask was charged with((2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydrownethyl)-2-(2-(2-methacrylamidoethoxy)ethoxy)tetrahydro-2H-pyran-3-yl)carbamicacid (272 mg, 0.72 mmol) (Formula 1505, prepared, for example, asdescribed in Example 17D), HPMA (211 mg, 1.47 mmol) (Formula 1506), adithio-pyridyl functionalized uRAFT agent of Formula 1601 where n is 4and R⁸ is CMP (12.5 mg, 0.0217 mmol), azobis(isobutyronitril) (0.98 mg,0.005 mmol), and 1.2 ml dimethylformamide. The reaction mixture wassubjected to four freeze-pump-thaw degassing cycles then stirred at 70°C. for 20 hours. The corresponding random polymeric product of Formula1602 (having about 30 W¹ groups and about 60 W² groups) was recovered byprecipitating the reaction mixture in acetone. Excess acetone wasremoved at reduced pressure to provide the random polymeric product,which was used without further purification.

19B. Formula 1c′ where X″ is Insulin-B, m is 1, n is 4, p is 90 (30W¹+60 W²), t is 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is2-hydroxypropyl

The Formula 1602 solution obtained in Example 19A (20 mg) was suspendedin 200 μl of dimethylformamide and added to an endotoxin-free tubecontaining Insulin-6 (1 mg) and stirred at room temperature for 3 hoursto afford the corresponding product of Formula 1c′(“F1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀”).The reaction mixture was then precipitated in acetone and purified fromthe reaction mixture via fast protein liquid chromatography (FPLC) usinga Superdex 200 prep grade column and used without further purification.

19C. Formula 1c′ where X″ is Insulin-B, m is 1, n is 4, p is 90 (30W¹+60 W²), t is 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is2-hydroxypropyl, using N-acetyl-D-galactosamine

By following the procedure of Examples 19A and 19B and substituting((2S,3S,4S,5S,6S)-4,5-dihydroxy-6-(hydroxymethyl)-2-(2-(2-methacrylamidoethoxy)ethoxy)tetrahydro-2H-pyran-3-yl)carbamicacid for Formula 1505, there was obtained the corresponding galactosylcompound of Formula 1c′(“F1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀”).

19D. Formula 1c′ where X″ is P31, m is 1, n is 4, p is 90 (30 W¹+60 W²),t is 1, R³ is NHAc, R⁴ is OH, R⁸ is CMP, and R¹⁰ is 2-hydroxypropyl

By following the procedure of Examples 19B and 19C and substituting 20mg of P31 for Insulin-B, there were obtained the corresponding glucosyland galactosyl compounds of Formula 1c′ where X″ is P31.

19E. Compounds of Formulae 1f′ and 1g′

By following the procedures of Examples 19A and 19B and substituting theuRAFT agent of Formula 1601 with a uRAFT agent of Formulae 600′ or 700′there are obtained the corresponding compounds of Formulae 601′ or 701′,which are in turn contacted with a compound of Formula 101′ to affordthe corresponding compound of Formula 1f or Formula 1g′, respectively.

Example 20 OT-1 Challenge-to-Tolerance Model

20A. As discussed above in Example 14, F1aA-OVA-m₄-n₈ andF1b-OVA-m₁-n₄-p₃₄ mitigated an OVA-specific immune response afteradjuvented OVA challenge.

20B. A total of 3×10⁵ CFSE-labeled OTI CD8+ T cells and 3×10⁵CFSE-labeled OTII CD4+ T cells were injected into CD45.1+ recipientmice. At 1 and 6 days following adoptive transfer, mice were i.v.administered saline solutions containing OVA,F1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀[“OVA-p(Gal-HPMA)”],F1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀[“OVA-p(Glu-HPMA)”], or saline alone. Each mouse treated withformulations containing OVA in its free or conjugated form, received themolar equivalent of 20 μg OVA. At 15 d following adoptive transfer, micewere challenged with 5 μg of OVA and 25 ng of ultrapure E. coli LPS(InvivoGen) in 25 μL of saline injected intradermally into each rear legpad (Hock method:total dose of 10 μg of OVA and 50 ng of LPS). Mice weresacrificed 4 days following challenge, and spleen and draining lymphnode cells were isolated for restimulation. For flow cytometry analysisof intracellular cytokines, cells were restimulated in the presence of 1mg/mL OVA or 1 μg/mL SIINFEKL peptide (Genscript) for 3 h. Brefeldin-A(5 μg/mL; Sigma) was added, and restimulation was resumed for anadditional 3 h before staining and flow cytometry analysis.

As shown in FIGS. 8A-8B, the administration of OVA-p(Gal-HPMA) andOVA-p(Glu-HPMA) resulted in significant reduction in the percentages ofOT-I cells (out of the total CD8+ T-cell population) and OT-II cells(out of the total CD4+ T-cell population). FIG. 8A shows thatOVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) administration significantly reducedOT-I cells as compared to mice receiving repeat administrations of OVAalone (e.g., unconjugated). Reduction was even greater when compared tomice receiving only OVA and LPS challenge (e.g., that received salineinjections). Notably, the reduction resulting from treatment withOVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) reduced OT-I cell levels to levelsnot significantly different from nave mice. Similarly, as shown in FIG.8B, OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) administration resulted insignificant reduction in OT-II cells as compared to mice receivingunconjugated OVA or challenge alone. These data indicate that theproduction of cells that are specifically designed to react whenencountering OVA as an antigen decreases, indicative of a reduction inimmune response to OVA.

Additionally, the administration of OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA)resulted in significant increases in antigen-specific regulatory T-cellsin the lymph node and spleen of mice. As shown in FIG. 9A, treatmentwith either of these conjugates induced significant increases inCD25+/FoxP3+ cells in the lymph node. Likewise, FIG. 9B showssignificant increases (vs. nave, challenge (saline alone), and OVAtreated animals) in CD25+/FoxP3+OT-II cells. These data indicate thatregulatory T cell production is upregulated, which in turn, indicatesthat the immune system is negatively modulated with respect to itsresponse to OVA (e.g., less responsive, or more tolerant).

Further building on the above data showing the increased tolerance to anantigen after delivery of that antigen complex with a liver targetingmoiety is the data shown in FIG. 10 . In this experiment, the percentageof cells expressing interferon gamma (IFNγ) was measured. IFNγ isproduced by CD4 and CD8 T cells after antigen-specific immunitydevelops. As shown in FIG. 10 , mice receiving only saline pre-challengehave approximately 60% of the total OTI cells expressing IFNγ. Incontrast, OVA-treated mice have about 40% IFNγ-expressing cells. Nearlythe same as nave mice, the OTI cells of OVA-p(Gal-HPMA) andOVA-p(Glu-HPMA)-treated mice have less than 20% IFNγ positive cells.This significant reduction in IFNγ indicates a reduction in themechanisms that drive antigen-specific immunity. Collectively, and inview of the additional disclosure herein, these data demonstrate thattargeting an antigen to the liver can reduce the antigen-specific immuneresponse to that antigen. In particular, targeting with glucose orgalactose results in significant shifts in the cell populationsresponsible for antigen-specific immunity, that shift demonstrating atolerance to the specific antigen.

20C. By following the procedures described in Example 20A or 20B andsubstituting the tested OVA compositions with other compositions ofFormula 1 followed by challenge with the unconjugated antigen X, thetreated animals demonstrate a tolerance to the specific antigen X.

Example 21 OTI/OTII Challenge to Tolerance Model

Using the model of Example 20, additionally with OTII cells (which areCD4⁺ T cells from CD45.2⁺ mice, analogous to the CD8⁺ T cell OTI cells),the ability ofF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀[“OVA-p(Gal-HPMA)”] andF1m′-OVA-m₁₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀[“OVA-p(Glu-HPMA)”] to induce T regulatory responses and preventsubsequent responses to vaccine-mediated antigen challenge weredemonstrated, moreover using different dosing regimens. 3×10⁵CFSE-labeled OTI and 3×10⁵ CFSE-labeled OTII cells were adoptivelytransferred to CD45.1⁺ mice (n=8 mice per group) on day 0. On days 1, 4and 7, tolerogenic doses or control doses were administered. In oneregimen, OVA was provided at a dose of 2.5 μg at day 1, 2.5 μg at day 4,and 16 μg at day 7. In another, OVA was provided at a dose of 7 μg atday 1, 7 μg at day 4, and 7 μg at day 7, for the same total dose.Likewise, pGal-OVA and pGlu-OVA were each administered in other groupsat the same dosings of 2.5 μg at day 1, 2.5 μg at day 4, and 16 μg atday 7 or 7 μg at day 1, 7 μg at day 4, and 7 μg at day 7, all dosesbeing on an OVA equivalent dose basis. In a final group, saline wasadministered on the same days. On day 14, the recipient mice were thenchallenged with OVA (10 μg) adjuvanted with lipopolysaccharide (50 ng)by intradermal injection. Characterization of the draining lymph nodeswas done on day 19, to allow determination as to whether or not deletionactually took place and whether regulatory T cells were induced from theadoptively transferred cells.

Profound tolerance was induced in the CD4+ T cell compartment, as shownin FIGS. 11A-11B. In terms of total cell frequencies, both dosingregimens of both OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) resulted inequivalent low levels of OTII cells after challenge, statistically lowerthan by treatment of OVA (* and # indicate p<0.05, ** and ## indicatep<0.01), as shown in FIG. 11A. When the cells that remained wereanalyzed by flow cytometry for the presence of the transcription factorFoxP3 and the receptor CD25, the numbers of FoxP3+CD25+ cells (markersof T regulatory cells) was statistically significantly elevated comparedto treatment with OVA alone, as shown in FIG. 11B. Here, the number of Tregulatory cells was statistically higher with the 2.5 μg/2.5 μg/16 μgdosing regimen compared to the 7 μg/7 μg/7 μg dosing regimen, with bothOVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) treatment.

Profound tolerance was also induced in the CD8+ T cell compartment, asshown in FIGS. 12A-12B. In terms of total cell frequencies, both dosingregimens of both OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) resulted inequivalent low levels of OTI cells after challenge, statistically lowerthan by treatment of OVA (* and # indicate p<0.05, ** and ## indicatep<0.01), as shown in FIG. 12A. When the cells that remained wereanalyzed by flow cytometry for the expression of IFN-γ after re-exposureto OVA antigen, the frequency of cells expressing this inflammatorycytokine was decreased in the groups receiving the 2.5 μg/2.5 μg/16 μgdosing regimen compared to the 7 μg/7 μg/7 μg dosing regimen, with bothOVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) treatment, as shown in FIG. 12B.

Example 22 BDC2.5 Study

22A. CD4+ T-cells of the transgenic NOD-BDC2.5 mice express thediabetogenic BDC-2.5 specific regulatory T-cell receptor (TCR). BDC2.5T-cells specifically target the islet beta-cell autoantigen,chromogranin-A. T-cells were isolated from the spleens of transgenicNOD-BDC2.5 mice and cultured for 4 days in DMEM supplemented with 10%(vol/vol) FBS, 0.05 mM β-mercaptoethanol, 1% puromycin/streptomycin, and0.5 μM P31 peptide, a mimotope of islet beta-cell autoantigenchromogranin-A that stimulates T-cells expressing the BDC2.5 T-cellreceptor. Following stimulation with P31, cells were washed with basalDMEM and analyzed for purity by flow cytometry, and 5×10⁶ T-cells werei.v. injected into normoglycemic NOD/ShiLtJ mice. At 8 h and 3 daysafter adoptive transfer, mice were i.v. administered saline, 10 μgF1c′-P31-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀, 10 μgF1c′-P31-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀, or anequimolar dose of P31 peptide. Starting on day 4, diabetes onset wasmonitored by measuring nonfasting blood glucose levels using anAccuCheck Aviva glucometer (Roche). Mice were considered diabetic atblood glucose readings ≥300 mg/dL. After two hyperglycemic readings,mice were euthanized. The data resulting from this experiment is shownin the time course of FIG. 13 . As shown, the mice receiving salinedeveloped diabetic blood glucose levels within 4-8 days of adoptivetransfer. Similarly, mice receiving P31 (unconjugated) developeddiabetic blood glucose levels within about 7-10 days after transfer. Instark contrast, mice receivingF1c′-P31-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀ orF1c′-P31-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ maintainedrelatively steady blood glucose values (<200 mg/dl) for over 40 days.

22B. By following the procedures described in Example 21A andsubstituting the tested compositions with other compositions of Formula1 where X is Insulin-6 or proinsulin, preproinsulin, glutamic aciddecarboxylase-65 (GAD-65 or glutamate decarboxylase 2), GAD-67,glucose-6 phosphatase 2 (IGRP or islet-specific glucose 6 phosphatasecatalytic subunit related protein), insulinoma-associated protein 2(IA-2), and insulinoma-associated protein 213 (IA-2(3), ICA69, ICA12(SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin-A,HSP-60, caboxypeptidase E, peripherin, glucose transporter 2,hepatocarcinoma-intestine-pancreas/pancreatic associated protein, S100β,glial fibrillary acidic protein, regenerating gene II, pancreaticduodenal homeobox 1, dystrophia myotonica kinase, islet-specificglucose-6-phosphatase catalytic subunit-related protein and SSTG-protein coupled receptors 1-5, such as F1aA-Insulin-m₂-n₈₀,F1aA-Insulin-m₂-n₁₂, F1aA-Insulin-m₂-n₃₃, F1aA-Insulin-m₂-n₄₀,F1aA-Insulin-m₂-n₄₃, F1aA-Insulin-m₂-n₅₀, F1aA-Insulin-m₂-n₆₀,F1aA-Insulin-m₂-n₇₅, F1aA-Insulin-m₂-n₈₄, F1b-Insulin-m₂-n₄-p₃₄-2NAcGAL,F1m-Insulin-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc, F1m-Insulin-m₂-n₆₂-p₃₀-q₈-CMP-2OH,F1n-insulin-m₂-n₁-p₃₀-q₄-CMP-2NHAc,F1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1 NAcGLU₃₀-ran-HPMA₆₀ orF1c′-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ theblood glucose values in the treated NOD mice remain steady as comparedto animals that receive saline.

Example 23 NOD Mouse

23A. Non-obese diabetic (NOD) mice, such as NOD/ShiLt mice aresusceptible to the spontaneous onset of autoimmune diabetes mellitus,which is the result of an autoimmune response to various pancreaticauto-antigens. Diabetes develops in NOD mice as a result of insulitis,characterized by the infiltration of various leukocytes into thepancreatic islets. As diabetes develops, there is a leukocyticinfiltration of the pancreatic islets followed by significant decreasesin insulin production, and corresponding increases in blood glucoselevels.

In order to evaluate the efficacy of a treatment for diabetes mellitus,compositions and methods for the treatment being provided in the presentdisclosure, starting at 5 weeks of age diabetes onset in a cohort ofNOD/ShiLt mice was monitored on a weekly basis by measuring nonfastingblood glucose levels using an AccuCheck Aviva glucometer (Roche).Starting at 6 weeks of age, the mice were divided into control and testgroups (n=15 for each group) and treated, respectively, with weeklyintravenous injections of saline, 10 μg ofF1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀, or 10μg of F1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀(10 μg). The injections continued for 10 consecutive weeks. Thepercentage of diabetes free animals was measured over time. Mice wereconsidered diabetic at two consecutive blood glucose readings ≥300 mg/dLor one blood glucose readings ≥450 mg/dL. Mice deemed diabetic wereeuthanized.

FIG. 14 depicts the data obtained as described above as the percentageof diabetes free animals as measured over time. Mice treated withF1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀ areshown as filled squares. Mice treated withF1C-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀ areshown as filled triangles. Mice treated with saline are shown as filleddiamonds. As can readily be appreciated from the data collected from thesaline treated animals over time as early as 11 weeks of age,spontaneous diabetes was present. Prevalence increased over time (shownby the downward trend in the graph) with 60% of the tested animalsdeveloping diabetes by week 20. As shown in FIG. 14 , treating NOD micewith either F1c′-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀ orF1c′-Insulin-B-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀reduced the incidences of diabetes onset in NOD mice as compared toanimals that received saline. The data demonstrate that administrationof insulin coupled with linkers and liver targeting moieties asdisclosed herein can successfully reduce the development of type Idiabetes mellitus by reducing the autoimmune response to the variouspancreatic autoantigens produced.

23B. By following the procedures described in Example 22A andsubstituting the tested compositions with other compositions of Formula1 where X is Insulin-6 or proinsulin, preproinsulin, glutamic aciddecarboxylase-65 (GAD-65 or glutamate decarboxylase 2), GAD-67,glucose-6 phosphatase 2 (IGRP or islet-specific glucose 6 phosphatasecatalytic subunit related protein), insulinoma-associated protein 2(IA-2), and insulinoma-associated protein 213 (IA-2(3), ICA69, ICA12(SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin-A,HSP-60, caboxypeptidase E, peripherin, glucose transporter 2,hepatocarcinoma-intestine-pancreas/pancreatic associated protein, S100β,glial fibrillary acidic protein, regenerating gene II, pancreaticduodenal homeobox 1, dystrophia myotonica kinase, islet-specificglucose-6-phosphatase catalytic subunit-related protein and SSTG-protein coupled receptors 1-5, such as F1aA-Insulin-m₂-n₈₀,F1aA-Insulin-m₂-n₁₂, F1aA-Insulin-m₂-n₃₃, F1aA-Insulin-m₂-n₄₀,F1aA-Insulin-m₂-n₄₃, F1aA-Insulin-m₂-n₅₀, F1aA-Insulin-m₂-n₆₀,F1aA-Insulin-m₂-n₇₅, F1aA-Insulin-m₂-n₈₄, F1b-Insulin-m₂-n₄-p₃₄-2NAcGAL,F1m-Insulin-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc, F1m-Insulin-m₂-n₆₂-p₃₀-q₈-CMP-2OH,F1n-insulin-m₂-n₁-p₃₀-q₄-CMP-2NHAc,F1c′-P31-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1 NAcGLU₃₀-ran-HPMA₆₀ orF1c′—P31-m₁-n₄-p₉₀-CMP-poly-(EtPEG₁AcN-1 NAcGAL₃₀-ran-HPMA₆₀ theincidences of diabetes onset in the treated NOD mice are reduced ascompared to animals that receive saline.

Example 24 Biodistribution

In order to examine the biodistribution of antigen glycopolymerconjugates we treated BALB/c mice with fluorescently labeled OVA orfluorescently-labeled OVA conjugated to either p(Gal-HPMA), p(Glu-HPMA),p(Galβ-HPMA), or p(Gluβ-HPMA). The sugar moieties attached to thebackbone of p(Gal-HPMA) and p(Glu-HPMA) are attached to the polymer inthe α-conformation at the C1 position, whereas the sugars attached tothe backbone of p(Galβ-HPMA) and p(Gluβ-HPMA) are attached to thepolymer in the β-conformation at the C1 position. OVA was labeled withDy750. All treatments were given via tail vein injection in 140 μl. Eachanimal was treated with an equal amount of fluorescent conjugate on afluorescence unit basis. After 3 hours, the animals were euthanized andthe livers of each animal were perfused with saline, then both thelivers and spleens were harvested and imaged via an IVIS Spectrum systemwith appropriate filter set.

FIG. 15 depicts representative images of the fluorescent signals oflivers (A) and spleens (B) from animals treated with OVA or OVAglycopolymer conjugates. The formulations are as follows: 1. OVA, 2. F1m′-OVA750-m₁₋₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1 NAcGALβ₃₀-ran-HPMA₆₀)[“OVA-p(Galβ-HPMA)”], 3.F1m′-OVA750-m₁₋₃-n₇₉-p₉₀-q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀)[“OVA-p(Gal-HPMA)”], 4. F1m′-OVA750-m₁₋₃-n₇₉-p₉₀-q₄-CMP-poly-(EtPEG₁AcN-1NAcGLUβ₃₀-ran-HPMA₆₀)[“OVA-p(Gluβ-HPMA)”], 5. F1m′-OVA750-m₁₋₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀)[“OVA-p(Glu-HPMA)”]. Images of the livers from animals treated asdescribed above show that glycopolymer conjugates significantly enhancethe delivery of their conjugated antigen to the liver (or spleen) ascompared to the uptake of unconjugated antigens. Livers from animalstreated with unconjugated OVA have less fluorescent signal as comparedto livers from animals treated with OVA conjugated to eitherp(Gal-HPMA), p(Glu-HPMA), p(Galβ-HPMA), or p(Gluβ-HPMA). Additionally,images of the spleens taken from animals treated as described above showthat conjugating antigens to glycopolymers reduces the delivery ofantigens to the spleen. Spleens from animals treated with unconjugatedOVA have significantly more fluorescent signal as compared to spleensfrom animals treated with OVA conjugated to either p(Gal-HPMA),p(Glu-HPMA), p(Galβ-HPMA), or p(Gluβ-HPMA). These data are significantin that they demonstrate enhanced targeting of an antigen to whichtolerance is desired to the liver and/or spleen, which, as demonstratedby the experimental data presented herein results in reduced immuneresponse (i.e., induced tolerance) to the antigen. In accordance withseveral embodiments disclosed herein, this induced tolerance can treat,reduce, prevent, or otherwise ameliorate an unwanted immune responsethat would have otherwise been associated with exposure to the antigen.

Example 25 7-Day OTI/OTII Phenotype Analysis

In order to compare the ability of various glycopolymer-antigenconjugates to induce antigen-specific T cell proliferation as well asupregulate the expression and presentation of various markers of T cellanergy and deletion, mice that had received an infusion of 400,000carboxyfluorescein succinimidyl ester (CSFE)-labeled OTI cells weretreated with an intravenous injection of either OVA or OVA conjugated toeither p(Gal-HPMA), p(Glu-HPMA), p(Galβ-HPMA), or p(Gluβ-HPMA) (withformulations as follows:F1m′-OVA-m₁₋₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGAL₃₀-ran-HPMA₆₀)[“OVA-p(Gal-HPMA)”]; F1m′-OVA-m₁₋₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLU₃₀-ran-HPMA₆₀)[“OVA-p(Glu-HPMA)”]; F1 m′-OVA-m₁₋₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGALβ₃₀-ran-HPMA₆₀) [“OVA-p(Galβ-HPMA)”]; F1m′-OVA-m₁₋₃-n₇₉-p₉₀q₄-CMP-poly-(EtPEG₁AcN-1NAcGLUβ₃₀-ran-HPMA₆₀)[“OVA-p(Gluβ-HPMA)”]. Animals treated with OVA in either its free orconjugated form received 10 μg of OVA on day 1 and day 3 of theexperiment. A timeline of the experimental details is shown in FIG. 16A.After 7 days, the mice were sacrificed and the splenocytes of theanimals were harvested and analyzed via flow cytometry for phenotypicalmarkers characteristic of T cell anergy, deletion, and memory.

FIG. 16B shows that OVA-glycopolymer conjugates induce more OTI T cellproliferation as compared to the amount of OTI proliferation seen inanimals treated with unconjugated OVA. As discussed above, these datafurther support that, according to several embodiments disclosed herein,the glyoctargeting moieties disclosed herein result in increasedantigen-specific T-cell proliferation a key step in inducing toleranceto an antigen. Interestingly, animals treated with OVA-glycopolymerconjugates containing β-linked sugars induced significantly moreproliferation compared to animals treated with glycopolymers containingthe same sugar moiety linked to the polymer via an α-linkage (e.g.,p(Galβ-HPMA) vs. p(Gal-HPMA)). Unexpectedly, this conformational changein one element of the overall composition leads to an enhanced efficacyin terms of T-cell proliferation, which Applicant believes (withoutbeing bound by theory) results from synergistic interaction of thecomponents of the composition with their respective physiologicaltargets. Additionally, the population of OTI cells taken from animalstreated with all OVA-glycopolymer conjugates, with the exception ofOVA-p(Gal-HPMA), showed significantly more surface expression of theapoptosis marker Annexin V+ as compared to the cells taken from animalstreated with OVA (see FIG. 16C). Consistent with data discussed above,this indicates a greater percentage of antigen-specific T cells arebeing targeted for, or are in, the apoptotic cascade. As shown in FIG.16D, OTI cells taken from animals treated with OVA-glycopolymerconjugates containing β-linked sugars showed an increased expression ofthe T cell exhaustion marker PD-1 as compared to animals treated withglycopolymers containing the same sugar moiety linked to the polymer viaan α-linkage as well as animals treated with free OVA. In order tomaintain long-term tolerance, treatments must reduce the number oflong-lasting antigen-specific memory T cells. FIGS. 16E and 16F showthat both OVA-p(Galβ-HPMA) and OVA-p(Gluβ-HPMA) induce a significantreduction in OTI cells expressing markers for memory T cells. BothOVA-p(Galβ-HPMA) and OVA-p(Gluβ-HPMA) induce a five-fold decrease in thenumber of memory T cells compared to animals treated with free OVA.These data further indicate that compositions as disclosed herein caninduce tolerance to an antigen (OVA chosen here due to its generalacceptance in the field as a “gold standard” antigen), and in severalembodiments, can unexpectedly enhance the induction of tolerance (asrepresented at least in part by antigen-specific T cell proliferation,increased Annexin V expression on antigen-specific T cells, increasedexhaustion marker expression on antigen-specific T cells, and reducedexpression of memory T cells).

Example 26 Glucose-Coupled Antigens—BDC2.5 Study

This experiment was conducted to test the ability of p(Glu)-antigenconjugates to inhibit the development of CD4 T cell driven autoimmunity. A mouse model of antigen-specific T cell induced autoimmunediabetes was used. Autoimmune diabetes was induced by transferringactivated BDC2.5 T cells, which carry a T cell receptor for the diabeticautoantigen chromogranin A, into immune compromised mice. Recipient micewere then treated with an autoantigen peptide mimotope, termed p31, orwith the p31-p(Glu) conjugates.

Protocol: Freshly isolated splenocytes from female BDC2.5 transgenicmice were stimulated for 4d in DMEM supplemented with 10% FBS, 0.05 mMβ-mercaptoethanol, 1% puromycin/streptomycin, and 0.5 μM p31 peptide.Following stimulation, cells were washed with DMEM, resuspended in DMEM,and injected intravenously into normoglycemic NOD/Scid mice. At 8 hfollowing adoptive transfer, mice were intravenously administered saline(nave), 2 μg of p31, or 2 μg of p31 as p31-pGlu conjugates (glucose inthe beta conformation was used in this experiment, though in severalembodiments the alpha conformation can be used). Three days after theinitial treatment, mice were treated again with saline, 2 μg of p31, or2 μg of p31 as p31-pGlu conjugates. Starting on day 1, the nonfastingblood glucose levels of each mouse were measured using an AccucheckAviva glucometer. Mice were considered diabetic upon receiving a singleblood sugar measurement over 450 mg/dL, or two consecutive blood sugarreadings of over 350 mg/dL. All groups had 7 mice.

Results: As shown in FIG. 17 , nave mice began to develop diabetes (asmeasured by hyperglycemia) within 5 days of the splenocyte transfer.Within about 7 days, all of the nave mice developed hyperglycemia. Thosemice treated with the control composition (p31; filled circles) began todevelop hyperglycemia shortly after the nave mice. By 8 dayspost-transfer, all of the p31-treated mice had developed hyperglycemia.In stark contrast, administration of p31-p(Glu) conjugates (filledtriangles) results in mice that maintain normal blood glucose levels fora significantly longer period of time as compared to nave animals andthose treated with the p31 peptide alone. At 15 days post-transfer, 100%of the p31-pGlu mice still had normal blood glucose levels.

These results demonstrate that p31-p(Glu) conjugates prevent thedevelopment of hyperglycemia in mice. Thus, according to severalembodiments disclosed herein, such conjugates are efficacious for use inpreventing development of diabetes. In still additional embodiments,p(Glu) conjugates are efficacious for use in reducing advancement ofpre-existing diabetes, and in still additional embodiments, efficaciousfor use in reversing pre-existing diabetes. In additional embodiments,other mimotopes of islet beta-cell autoantigen chromogranin-A can beused. Further, chromogranin-A, as a full-length protein, or fragmentsthereof can be employed as the antigenic portion of the tolerogeniccompositions, according to several embodiments disclosed herein.Additionally, other antigens disclosed herein related to diabetes can beused, in some embodiments, including but not limited to insulin,proinsulin, preproinsulin, GAD-65, GAD-67, IGRP, IA-2, IA-2β, fragmentsthereof, or mimotopes thereof. Other diabetes related antigens aredisclosed herein. Further, in several embodiments, antigens related toother autoimmune diseases can be used, as is disclosed herein. In stilladditional embodiments, foreign antigens, transplant antigens or othertypes/classifications may be used, as chromogranin A was employed here,and using this model as a non-limiting example of the ability ofcompositions as disclosed herein to be used in the induction oftolerance.

Example 27 Memory of Tolerance

The following experiment was conducted to demonstrate that tolerogeniccompositions as disclosed herein can establish memory of tolerance. Inorder to determine if regulatory T cells (Tregs) induced byadministration of tolerogenic compositions as disclosed herein(p(Glu)-antigen conjugates were used here as a non-limiting example) areable to establish long term tolerance after the initial administrationof the therapies, mice were pre-treated with an infusion of OTII T cellsfollowed by administration of p(Glu)-OVA conjugates. These mice werethen were challenged three weeks later with an infusion of OVA-specificT cells and antigen challenge. To demonstrate that any toleranceinducing effect was the result of Tregs, mice treated with p(Glu)-OVAwere injected with an anti-CD25 antibody, which has been shown todeplete Tregs.

Protocol: On day zero, C57BL/6 mice received an intravenous infusion of450,000 OTII cells, then were treated on days 1 and 4 with saline or 1.5μg of OVA as free OVA or p(Glu)-OVA conjugates (OVA is used herein as anon-limiting example of an antigen to which tolerance is desired;glucose in the beta conformation was used in this experiment, though inseveral embodiments the alpha conformation can be used). On day 7, micereceived a final treatment of saline, or 15 μg of OVA as free OVA orp(Glu)-OVA conjugates. On day 15, half of the animals treated withp(Glu)-OVA were treated with an intraperitoneal injection of 300 μg ofanti-CD25 antibody, which has been shown to deplete regulatory T cells(Tregs). On day 29, mice received an infusion of 750,000 OTII T cellsand 750,000 OTI T cells. The next day, animals were challenged with 5 μgof OVA and 50 ng of ultrapure LPS in each of the 4 footpads. Five daysafter antigen challenge, the mice were sacrificed and the number of OTIand OTII T cells in the draining lymph nodes was assessed by flowcytometry. (Groups n=5: Challenge (i.e. vehicle treated animals), OVA,p(Glu)-OVA, p(Glu)-OVA+αCD25). This protocol is schematically depictedin FIG. 18 .

Results: The results of this experiment are shown in FIGS. 19A-19B. FIG.19A depicts the remaining OTI T cells after antigen challenge (as apercentage of total CD8⁺ cells). FIG. 19B depicts the remaining OTII Tcells after antigen challenge (as a percentage of total CD4⁺ cells).Those mice receiving LPS challenge and OVA absent a tolerogeniccomposition showed significantly elevated OTI and OTII cells in draininglymph nodes. Those mice receiving pGlu-OVA showed a significantreduction in both OTI and OTII cells. The abrogation of the toleranceinduced by pGlu-OVA by the administration of anti-CD25 antibodiesdemonstrates that there is a role for Tregs in the pGlu-OVA-inducedtolerance. Taken together, these data indicate that the tolerogeniccompositions disclosed herein are able to induce long-lasting tolerancethrough the induction of regulatory T cells. This is advantageous, inseveral embodiments, because the long-lasting tolerance reduces (or insome embodiments eliminates) the need for ongoing administration of thecompositions disclosed herein. That being said, in some embodiments,repeated administration is optionally performed.

Example 28 Memory of Tolerance (Endogenous)

The following experiment was conducted to demonstrate that tolerogeniccompositions as disclosed herein can establish memory of tolerance fromendogenous T cells. This experiment was conducted similarly to Example27, but the mice did not receive the initial infusion of donor OTII Tcells.

In order to determine if Tregs from the endogenous T cell populationcould be induced by p(Glu)-antigen conjugates and exhibit long-termtolerance, mice were treated with p(Glu)-OVA conjugates. They were thenchallenged three weeks later with an infusion of OVA-specific T cellsand an antigen challenge. To investigate whether the demonstratedtolerance inducing effect was the result of endogenous Tregs, mice weretreated with p(Glu)-OVA and later treated with anti-CD25 antibody.

Protocol: On day zero and 3, BL6/C57 mice received an intravenousinfusion of saline or 1.5 μg of OVA as free OVA or p(Glu)-OVA conjugates(glucose in the beta conformation was used in this experiment, though inseveral embodiments the alpha conformation can be used). On day 6, micereceived a final treatment of saline, or 15 μg of OVA as free OVA orp(Glu)-OVA conjugates. On day 14, half of the animals treated withp(Glu)-OVA were treated with an intraperitoneal injection of 300 μg ofanti-CD25 antibody. On day 28, mice received an infusion of 750,000 OTIIand 750,000 OTI T cells. The next day, animals were challenged with 5 μgof OVA and 50 ng of ultrapure LPS in each of the 4 footpads. Five daysafter antigen challenge, the mice were sacrificed and the number of OTIand OTII T cells in the draining lymph nodes was assessed by flowcytometry. (Groups n=5: Challenge (i.e. vehicle treated animals), OVA,p(Glu)-OVA, p(Glu)-OVA+αCD25). This protocol is schematically depictedin FIG. 20 .

Results: The results of this experiment are shown in FIGS. 21A-21B. FIG.21A depicts the remaining OTI T cells after antigen challenge (as apercentage of total CD8⁺ cells). FIG. 21B depicts the remaining OTII Tcells after antigen challenge (as a percentage of total CD4⁺ cells).Those mice receiving LPS challenge and OVA absent a tolerogeniccomposition showed significantly elevated OTI and OTII cells in draininglymph nodes. Those mice receiving pGlu-OVA showed a significantreduction in both OTI and OTII cells in comparison. The abrogation ofthe tolerance induced by pGlu-OVA by the administration of anti-CD25antibodies demonstrates that Tregs derived from the endogenous T cellpopulation play a significant role in the tolerance induction by thepGlu-antigen composition. Taken together, these data indicate that thetolerogenic compositions disclosed herein are able to inducelong-lasting tolerance through the induction of endogenous regulatory Tcells. This is advantageous, in several embodiments, because thelong-lasting tolerance reduces (or in some embodiments eliminates) theneed for ongoing administration of the compositions disclosed herein.That being said, in some embodiments, repeated administration isoptionally performed. Moreover, these data indicate that supplementingthe T cells of a recipient with exogenous T cells is not necessary forinduction of tolerance. Rather, the endogenous pool of T cells issufficient to provide regulatory T cells in appropriate numbers toresult in long-term tolerance.

Example 29 Prophylactic Administration of Tolerogenic CompositionsReduces Subsequent Antibody Generation

As disclosed herein, in several embodiments, the tolerogeniccompositions are useful for the reduction, treatment, prevention orotherwise amelioration of immune responses against antigens of interest.In some embodiments, therefore the ultimate use of the tolerogeniccomposition, e.g., prevention versus treatment, is determined by thecurrent state of a subject prior to receiving administration of thecomposition. The present experiment was performed in order todemonstrate that tolerogenic compositions disclosed herein can be usedto reduce the degree of antibody generation to a specific antigen,through the prophylactic administration of a tolerogenic composition.

Protocol: The experimental design is depicted in FIG. 22 . Forpretreatment, there were two groups, those receiving 15 μg of atolerogenic composition comprising p-Glu-Asparaginase (high dose) andthose receiving 2.5 μg of p-Glu-Asparaginase (low dose) (glucose in thebeta conformation was used in this experiment, though in severalembodiments the alpha conformation can be used). These pretreatmentsteps are depicted in FIG. 22 as step “A”. Note that, asparaginase isused in this experiment as a nonlimiting example of an antigen to whichtolerance is desired. As discussed above, numerous other antigens,fragments thereof, or mimotopes thereof can also be used, depending onthe embodiment. After the pretreatment, or at the initiation of theexperimental protocol for the wild type asparaginase (WT-Asn) and mixedgroups, animals were administered either 15 μg of asparaginase or acombination of 12.5 μg of asparaginase along with 2.5 μg ofpGlu-Asparaginase. These administrations are depicted as step “B” inFIG. 22 . For all experimental groups, step “C” represents collection ofa 10 μL blood sample.

Results: results of this experiment are shown in FIG. 23 . The tracethat show increases in anti-asparaginase antibody titers beginning atapproximately two days and steadily increasing to relative values ofbetween 3 and 4 are the result of the administration of asparaginasealone. In contrast, the pretreatment with either high dose or low doseasparaginase significantly reduced the generation of anti-asparaginaseantibodies, and consistently held the antibody titer at relatively lowervalues through the 59 day testing protocol. The negative control group(antigen nave) shows no development of antigen-specific antibodies.

These data demonstrate that, in addition to treating or reducing apre-existing immune response to an antigen of interest, in severalembodiments, the tolerogenic compositions disclosed herein can also beused in preventing the initial immune response. As shown in thisexperiment, both high and low doses of glycotargeting therapeuticscoupled to an antigen of interest administered as a pretreatmentameliorated the generation of antibodies against the antigen ofinterest. In several embodiments, such an approach is particularlyeffective when an exogenous therapeutic agent is to be administered to apatient. In some such embodiments depending upon the therapeutic agent,the tolerogenic composition need not include the entire therapeuticagent as the antigen of interest (although in some embodiments theentire therapeutic agent is included), but rather can include a fragmentof the therapeutic agent a particular epitope of the therapeutic agent,or a mimotope of an antigenic region of the therapeutic agent. In someembodiments, the therapeutic agent is a protein drug. Additionally, insome embodiments longer and/or more frequent pretreatmentadministrations of the tolerogenic composition may serve to even furtherreduce the generation of antibodies against the antigen of interest.However, in some embodiments a single pretreatment step may besufficient.

Example 30 Tolerogenic Compositions Ameliorate Multiple Sclerosis

As discussed above, a variety of diseases associated with an immuneresponse, including autoimmune responses, can be treated through thegeneration and use of tolerogenic compositions as disclosed herein. As anonlimiting example of such a used in the autoimmune context, thepresent experiment was designed to determine the efficacy of atolerogenic composition in the treatment of multiple sclerosis (MS).

The MS-related tolerogenic compositions were tested in a mouse model ofmultiple sclerosis (experimental autoimmune encephalomyelitis, EAE). Theauto-antigen used for vaccination in the model was a peptide derivedfrom myelin oligodendrocyte glycoprotein (MOG), amino acid numbers 35-55(MOG35-55; SEQ ID NO:24). The auto-antigen utilized for treatment in themodel was a slightly longer peptide sequence (MOG30-60; SEQ ID NO:25),which contained the vaccination peptide specified above. The longertherapeutic sequence was used to ensure processing by antigen presentingcells and consequently reduce the tendency of the soluble peptide tobind to cell-surface major histocompatibility complex in the absence ofuptake by antigen presenting cells.

Protocol: The experimental protocol is shown in FIG. 24A, with thetherapeutic tolerogenic composition shown in FIG. 24B. EAE was inducedby immunizing donor B6.SJL mice with MOG35-55/CFA. 11 days later, theirspleen cells are harvested and restimulated in culture with MOG35-55peptide, anti-IFNγ antibodies and IL-12 for 3 days. The resultantencephalitogenic cells were injected into recipient groups of mice,which then develop MS symptoms. Group sizes were 10 mice each, of whichhalf were sacrificed at the peak of disease (Day 11) and half weresacrificed at the end of experiment (Day 20). Body weights were measured3 times per week and disease state was scored daily, blinded. Controlpeptide (MOG30-60), glycotargeting peptide (pGlu-MOG30-60; (glucose inthe alpha conformation was used in this experiment, though in severalembodiments the beta conformation can be used), or vehicle (saline) weregiven on Days 0, 3, and 6 in either 0.8 μg or 4.0 μg doses. A positiveefficacy treatment control (FTY720) was given daily for the duration ofthe study. FTY720 (fingolimod) is a first-in-class sphingosine1-phosphate (SIP) receptor modulator deemed effective in Phase IIclinical trials for MS.

FIG. 25A shows the assessment of the tolerogenic composition's abilityto delay disease onset as compared to control animals. As can be seen,at day 11 (the peak of the disease symptoms for the control group),administration of 0.8 μg of pGlu-MOG₃₀₋₆₀ (filled triangles) resulted ina significant decrease in the EAE disease score (as compared to the MOGpeptide alone). FIG. 25B shows data in relation to reduction of weightloss that is associated with MS. Similar to the EAE disease score,pGlu-MOG₃₀₋₆₀ showed significantly reduced weight loss at day 11 of theexperiment (FIG. 25B; as compared to MOG alone). In contrast, thepeptide alone group and the control group exhibited loss ofapproximately 25% of body weight at the same time point in theexperiment.

FIG. 26A corresponds to the assessment of disease onset delay with thehigher, 4 μg dose of pGlu-MOG₃₀₋₆₀. Unexpectedly, the delay in diseaseonset was strikingly improved (significant vs. both MOG alone andFTY720, with none of the mice in the treatment group (filled triangles)exhibiting a nonzero EAE disease score until 14 days into theexperiment. In contrast, by day 6, each of the other experimental groupswas showing signs of MS, as evidenced by the increased EAE diseasescore. Again, similar to EAE disease score, the higher, 4 μg dose ofpGlu-MOG₃₀₋₆₀ resulted in significant reductions in weight loss. At day11, the pGlu-MOG₃₀₋₆₀ group (closed triangles) had essentially zeroweight loss, while all other groups had lost weight (˜2.5 grams in theFTY720 group and ˜5 grams in the MOG alone and control groups, see FIG.26B). Thus, the higher, 4 μg dose of pGlu-MOG₃₀₋₆₀ yielded significantly(vs. both MOG alone and FTY720) improved retention of weight, whichlasted throughout substantially all of the experiment.

As discussed above, this particular experiment was a nonlimiting exampleof how a tolerogenic composition according to several embodimentsdisclosed herein could be used to reduce the immune response that asubject generates to an antigen of interest, here an antigen associatedwith the autoimmune disease multiple sclerosis. As mentioned previously,other antigens, autoantigens, therapeutic proteins, fragments orspecific epitopes of any of the preceding, or mimotope of any of thepreceding can also successfully be used in accordance with thedisclosure provided herein in order to induce immune tolerance.

Example 31 Biodistribution of pGal and pGlu Beta Conjugates

This experiment was conducted to determine the hepatic cell typestargeted by pGal and pGlu conjugates as disclosed herein.

Protocol: mice were treated via tail vein injection with 100 μg of OVAfluorescently labeled with the fluorescent dye Dy-649 (OVA649), 100 μgof OVA conjugated to pGluβ (OVA649-pGluβ), or 100 μg of OVA conjugatedto pGalβ (OVA649-pGluβ). After 3 h, these mice were sacrificed and thelivers were harvested, processed into single cell suspensions, andseparated via density gradient centrifugation. The various hepatic celltypes were then analyzed for protein content (e.g., as a measure ofOVA649) via flow cytometry.

Results: Results show that both OVA649-pGalβ and OVA649-pGluβ conjugatesare more effective at targeting liver sinusoidal endothelial cells(LSECs) as compared to OVA649 (FIG. 27A). LSECs taken from animalstreated with OVA649-pGalβ had a two-fold increase in mean fluorescentintensity (MFI) and LSECs taken from animal treated with OVA649-pGluβhad a 2.5-fold increase in MFI as compared to LSECs taken from animalstreated with OVA649.

Kupffer Cells were also efficiently targeted by OVA649-pGluβ conjugates.The percentage of Kupffer cells that took up OVA649-pGluβ in animalstreated with OVA649-pGluβ was significantly greater than the percentageof Kupffer Cells that took up OVA649 (FIG. 27B). In several embodiments,pGlu in the beta conformation is therefore used when it may be desirableto target Kupffer cells. That being said, pGal conjugates can alsooptionally be used in certain embodiments.

In addition to Kupffer cells, CD11c+ cells also efficiently took upOVA649-pGluβ. The percentage of CD11c+ cells that took up OVA649-pGluβwas significantly greater than the percentage of CD11c+ that weretargeted by OVA649 (FIG. 27C). In several embodiments, pGlu in the betaconformation is therefore used when it may be desirable to target CD11C+cells. That being said, pGal conjugates can also optionally be used incertain embodiments.

Interestingly, both OVA649-pGluβ and OVA649-pGalβ conjugates were moreeffective at targeting hepatocytes as compared to OVA649. See FIG. 27D.However, the percentage of hepatocytes that took up OVA649-pGalβ wasgreater than the percentage of hepatocytes targeted by OVA649-pGluβ.Thus, in several embodiments, pGal in the beta conformation is thereforeused when it may be desirable to target hepatocytes. That being said,pGlu conjugates can also optionally be used in certain embodiments.

Interestingly, an analysis of the ability of free OVA andOVA-glycopolymer conjugates to target stellate cells showed contrastingresults. FIG. 27E shows that OVA targets stellate cells more efficientlythan either of the OVA-glycopolymer conjugates.

In several embodiments, combinations of pGlu and pGal conjugates may bedesirable and the two types of composition interact synergistically totarget the liver (and various cell types in the liver). Also, in severalembodiments mixtures of conjugates in the alpha and beta configurationscan also be used. As with other experiments disclosed herein, the use ofOVA is as a non-limiting example of an antigen to which tolerance isdesired. Other antigens disclosed herein, fragments thereof, andmimotopes thereof can also be conjugated to glycotargeting moieties, andtolerance thereto can be induced, based on the disclosure providedherein. In accordance with several embodiments disclosed herein, thisinduced tolerance can treat, reduce, prevent, or otherwise ameliorate anunwanted immune response that would have otherwise been associated withexposure to the antigen.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes can be made and equivalents can besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications can be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the claims appended hereto. All publications, patents, patentapplications, internet sites, and accession numbers/database sequences(including both polynucleotide and polypeptide sequences) cited areherein incorporated by reference in their entirety for all purposes tothe same extent as if each individual publication, patent, patentapplication, internet site, or accession number/database sequence werespecifically and individually indicated to be so incorporated byreference.

It is contemplated that various combinations or subcombinations of thespecific features and aspects of the embodiments disclosed above may bemade and still fall within one or more of the inventions. Further, thedisclosure herein of any particular feature, aspect, method, property,characteristic, quality, attribute, element, or the like in connectionwith an embodiment can be used in all other embodiments set forthherein. Accordingly, it should be understood that various features andaspects of the disclosed embodiments can be combined with or substitutedfor one another in order to form varying modes of the disclosedinventions. Thus, it is intended that the scope of the presentinventions herein disclosed should not be limited by the particulardisclosed embodiments described above. Moreover, while the invention issusceptible to various modifications, and alternative forms, specificexamples thereof have been shown in the drawings and are hereindescribed in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner; however, they can also include any third-party instructionof those actions, either expressly or by implication. For example,actions such as “administering a glycotargeting tolerogenic composition”include “instructing the administration of a glycotargeting tolerogeniccomposition.” In addition, where features or aspects of the disclosureare described in terms of Markush groups, those skilled in the art willrecognize that the disclosure is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between,” and the like includesthe number recited. Numbers preceded by a term such as “about” or“approximately” include the recited numbers. For example, “about 10nanometers” includes “10 nanometers.”

What is claimed is:
 1. A composition for the induction ofantigen-specific immune tolerance in a subject, the compositioncomprising: an antigen to which tolerance is desired; wherein theantigen to which tolerance is desired is capable of inducing an unwantedimmune response in the subject; a liver targeting moiety; and apolymeric linker comprising:

where the right bracket “)” indicates a bond between the linker and acorresponding reversible addition-fragmentation chain transfer (RAFT)polymerization agent; the bottom bracket “

” indicates a bond between the linker and the liver targeting moiety; Wis a copolymer or a random copolymer of the W¹ and W² having p repeatunits, where:

where: p is an integer from 2 to 150; R⁹ is a direct bond,—C(O)—NH—CH₂—CH₂—, or —C(O)—NH—(CH₂—CH₂—O—)_(t)—CH₂—CH₂—; t is aninteger from 1 to 5; R¹⁰ is an aliphatic group, an alcohol or analiphatic alcohol; Y′ comprises:

where n is an integer from 1 to 100; and the left bracket “(” of Y′indicates the bond between the antigen and linker; the right bracket “)”of Y′ indicates a bond between Y′ and the remainder of the polymericlinker; wherein the polymeric linker is bonded to the antigen to whichtolerance is desired via a disulfide bond or a disulfanyl ethyl ester,wherein the disulfide bond or the disulfanyl ethyl ester are eachconfigured to cleave after administration of the composition to thesubject and to release the antigen to which tolerance is desired fromthe polymeric linker; and wherein the liver-targeting moiety comprises agalactosylating moiety.
 2. The composition of claim 1, wherein the livertargeting moiety is a beta anomer, wherein the liver targeting moietycomprises N-acetylgalactosamine, wherein the N-acetylgalactosamine isconjugated at its C1, C2 or C6 carbon to the polymeric linker, andwherein the antigen is associated with Celiac disease.
 3. Thecomposition of claim 1, wherein the antigen which tolerance is desiredis associated with Celiac disease and comprises one or more of SEQ IDNO: 21, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO:
 23. 4. Thecomposition of claim 1, further comprising one or more additionalantigens.
 5. The composition of claim 1, wherein the antigen to whichtolerance is desired is associated with Celiac disease and comprises atleast a portion of SEQ ID NO:
 21. 6. The composition claim 4, whereincompound further comprises an additional antigen to which tolerance isdesired, wherein the additional antigen is a food antigen.
 7. Acomposition for the induction of antigen-specific immune tolerance in asubject, the composition comprising: an antigen to which tolerance isdesired; wherein the antigen to which tolerance is desired is capable ofinducing an unwanted immune response in the subject; a liver-targetingmoiety; wherein the liver-targeting moiety comprises a galactosylatingor glucosylating moiety; a polymeric linker comprising:

where the right bracket “)” indicates a bond between the linker and acorresponding reversible addition-fragmentation chain transfer (RAFT)polymerization agent; the bottom bracket “

” indicates a bond between the linker and a liver targeting moiety; W isa copolymer or a random copolymer of the W¹ and W² having p repeatunits, where:

where: p is an integer from 2 to 150; R⁹ is a direct bond,—C(O)—NH—CH₂—CH₂—, or —C(O)—NH—(CH₂—CH₂—O—)_(t)—CH₂—CH₂—; t is aninteger from 1 to 5; R¹⁰ is an aliphatic group, an alcohol or analiphatic alcohol; Y′ comprises: where

n is an integer from 1 to 100; and the left bracket “(” of Y′ indicatesthe bond between the antigen and linker; the right bracket “)” of Y′indicates a bond between Y′ and the remainder of the polymeric linker;wherein the polymeric linker is bonded to the antigen to which toleranceis desired via a disulfide bond or a disulfanyl ethyl ester, wherein thedisulfide bond or the disulfanyl ethyl ester are each configured tocleave after administration of the compound to the subject and torelease the antigen to which tolerance is desired from the polymericlinker.
 8. The composition of claim 7, further comprising one or moreadditional antigens.
 9. The composition of claim 7, wherein the antigento which tolerance is desired is a food antigen.
 10. The composition ofclaim 7, wherein the antigen to which tolerance is desired comprises oneor more portions of deamidated alpha gliadin, native alpha gliadin andomega gliadin.
 11. The composition of claim 7, wherein the antigen towhich tolerance is desired comprises at least one tolerogenic portion ofalpha gliadin.
 12. The composition of claim 7, wherein the antigen towhich tolerance is desired comprises an amino acid sequence of SEQ IDNO: 21, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO:
 23. 13. Thecomposition of claim 7, wherein the antigen to which tolerance isdesired is associated with Celiac disease.
 14. The composition of claim13, wherein the antigen to which tolerance is desired comprises SEQ IDNO:
 21. 15. The composition claim 14, further comprising SEQ ID NO: 20.16. The composition of claim 7, wherein the antigen to which toleranceis desired is a food antigen selected from portions of one or more ofhigh molecular weight glutenin, low molecular weight glutenin, alpha-,gamma- and omega-gliadin, hordein, secalin and/or avenin.
 17. Acomposition for the induction of antigen-specific immune tolerance in asubject, the composition comprising: one or more antigens to whichtolerance is desired, wherein the one or more antigens to whichtolerance is desired is capable of inducing an unwanted immune responsein the subject; a liver targeting moiety, wherein the liver targetingmoiety comprises N-acetylgalactosamine; a polymeric linker:

where the right bracket “)” indicates a bond between the linker and acorresponding reversible addition-fragmentation chain transfer (RAFT)polymerization agent; the bottom bracket “

” indicates a bond between the linker and a liver targeting moiety; W isa copolymer or a random copolymer of the W¹ and W² having p repeatunits, where:

where: p is an integer from 2 to 150; R⁹ is a direct bond,—C(O)—NH—CH₂—CH₂—, or —C(O)—NH—(CH₂—CH₂—O—)_(t)—CH₂—CH₂—; t is aninteger from 1 to 5; R¹⁰ is an aliphatic group, an alcohol or analiphatic alcohol; Y′ comprises: where

n is an integer from 1 to 100; and the left bracket “(” of Y′ indicatesthe bond between the antigen and linker; the right bracket “)” of Y′indicates a bond between Y′ and the remainder of the polymeric linker;wherein the polymeric linker is bonded to the antigen via a disulfidebond or a disulfanyl ethyl ester, and wherein the disulfide bond or thedisulfanyl ethyl ester are each configured to cleave afteradministration of the compound to the subject and to release the antigenfrom the polymeric linker.
 18. The composition of claim 17, wherein theone or more antigens to which tolerance is desired comprises one or moreof SEQ ID NO: 21 or SEQ ID NO:
 20. 19. The composition of claim 17,wherein the one or more antigens to which tolerance is desired isassociated with Celiac disease.
 20. The composition of claim 17, whereinthe one or more antigens to which tolerance is desired comprises anamino acid sequence of SEQ ID NO: 21.